In 1525, Henry VIII became enamoured of Anne and began pursuing her. She resisted all his attempts to seduce her, refusing to become his mistress as her sister, Mary Boleyn, had. It soon became the one absorbing object of Henry's desires to annul his marriage to Queen Catherine, so he would be free to marry Anne. When it became clear that Pope Clement VII would not annul the marriage, the breaking of the power of the Catholic Church in England began.

The Archbishop of York, Thomas Wolsey, was dismissed to his diocese, allegedly at Anne Boleyn's instigation, and later the Boleyn family's chaplain, Thomas Cranmer, was appointed as Archbishop of Canterbury. Henry and Anne married on 25 January 1533. On 23 May 1533, Cranmer declared Henry and Catherine's marriage null and void; five days later, he declared Henry and Anne's marriage to be good and valid. Shortly afterwards, the Pope decreed sentences of excommunication against Henry and Cranmer. As a result of this marriage and these excommunications, the first break between the Church of England and Rome took place, and the Church of England was brought under the King's control.

Anne was crowned Queen of England on 1 June 1533. On 7 September, she gave birth to the future Elizabeth I of England. To Henry's displeasure, however, she failed to produce a male heir. Henry was not totally discouraged, for he said that he loved Elizabeth and that a son would surely follow. Three miscarriages followed, however, and by March 1536, Henry was courting Jane Seymour. In April-May 1536, Henry had Anne investigated for high treason. On 2 May, she was arrested and sent to the Tower of London, where she was tried before a jury of peers and found guilty on 15 May. She was beheaded four days later on Tower Green. Modern historians view the charges against her, which included adultery and incest, as unconvincing. Following the coronation of her daughter, Elizabeth, as queen, Anne was venerated as a martyr and heroine of the English Reformation, particularly through the works of John Foxe. Over the centuries, she has inspired or been mentioned in numerous artistic and cultural works. As a result, she has retained her hold on the popular imagination. Anne has been called "the most influential and important queen consort England has ever had," since she provided the occasion for Henry VIII to divorce Catherine of Aragon, and declare his independence from Rome.

Anne was the daughter of Thomas Boleyn, later Earl of Wiltshire and Earl of Ormond, and his wife, Lady Elizabeth Howard, daughter of Thomas Howard, 2nd Duke of Norfolk. Thomas Boleyn was a respected diplomat with a gift for languages; he was also a favourite of Henry VII of England, who sent him on many diplomatic missions abroad. Anne and her siblings grew up at Hever Castle in Kent. A lack of parish records from the period has made it impossible to establish Anne's date of birth. Contemporary evidence is contradictory, with several dates having been put forward by various historians. An Italian, writing in 1600, suggested that she had been born in 1499, while Sir Thomas More's son-in-law, William Roper, indicated a much later date of 1512. However her birth was most likely sometime between 1501 and 1507. As with Anne herself, it is uncertain when her two siblings were born, but it seems clear that her sister Mary was older than Anne. Mary's children clearly believed their mother had been the elder sister. Most historians now agree that Mary was born in 1499. Mary's grandson claimed the Ormonde title in 1596 on the basis she was the elder daughter, which Elizabeth I accepted. Also, Mary was married first, and by custom, the eldest daughter would always be married before the younger. Their brother George was born some time around 1504.

The academic debate about Anne's birth date focuses on two key dates: 1501 and 1507. Eric Ives, a British historian and legal expert, advocates the 1501 date, while Retha Warnicke, an American scholar who has also written a biography of Anne, prefers 1507. The key piece of surviving written evidence is a letter Anne wrote sometime in 1514. She wrote it in French to her father, who was still living in England while Anne was completing her education in the Netherlands. Ives argues that the style of the letter and its mature handwriting prove that Anne must have been about thirteen at the time of its composition, while Warnicke argues that the numerous misspellings and grammar errors show that the letter was written by a child. In Ives's view, this would also be around the minimum age that a girl could be a Maid of Honour, as Anne was to the regent, Margaret of Austria, Duchess of Savoy. This is supported by claims by a chronicler from the late 16th century, who wrote that Anne was twenty when she returned from France. These findings are contested by Warnicke in several books and articles, but the evidence does not conclusively support either date.

Anne's great-grandparents included a Lord Mayor of London, a duke, an earl, two aristocratic ladies, and a knight. One of them, Geoffrey Boleyn, had been a mercer and wool merchant before becoming Lord Mayor. The Boleyn family originally came from Blickling in Norfolk, fifteen miles north of Norwich. At the time of Anne's birth, the Boleyn family was considered one of the most respected in the English aristocracy. Among her relatives, she numbered the Howards, one of the pre-eminent families in the land. She was certainly of more noble birth than Jane Seymour, Henry VIII's later wife. The spelling of the Boleyn name was variable. Sometimes it was written as Bullen, hence the bull heads which formed part of her family arms. At the court of Margaret of Austria in the Netherlands, Anne is listed as Boullan. From there she signed the letter to her father as Anna de Boullan. She is also referred to as "Anna Bolina" (which is Latin); that name is in most portraits of her.

Anne's father continued his diplomatic career under Henry VIII. In Europe, Thomas Boleyn's charm won many admirers, including Archduchess Margaret of Austria, daughter of Maximilian I, Holy Roman Emperor. During this period, she ruled the Netherlands on her father's behalf and was so impressed with Boleyn that she offered his daughter Anne a place in her household. Ordinarily, a girl had to be twelve years old to have such an honour, but Anne may have been younger, as the Archduchess affectionately referred to her as "la petite Boulin [sic]". Anne made a good impression in the Netherlands with her manners and studiousness, Margaret reported that she was well spoken and pleasant for her young age ("son josne eaige"). and told Sir Thomas Boleyn that his daughter was "so presentable and so pleasant, considering her youthful age, that I am more beholden to you for sending her to me, than you to me" (E.W. Ives, op.cit.). Anne stayed with Margaret from spring 1513 until her father arranged for her to attend Henry VIII's sister, Mary Tudor, Queen of France, for Mary's marriage to Louis XII of France in October 1514.

In France, Anne was a maid of honour to Queen Mary, and then to 15-year-old Queen Claude of France, with whom she stayed nearly seven years. In the Queen's household, she completed her study of French and developed interests in fashion and religious philosophy. She also acquired knowledge of French culture and etiquette. Though all knowledge about Anne's experiences in the French court are conjecture, even Eric Ives, in his latest edition of the definitive biography, conjectures that she was likely to have made the acquaintance of King Francis I's sister, Marguerite de Navarre, a patron of humanists and reformers. Marguerite de Navarre was also an author in her own right, and her works include elements of Christian mysticism and reform that, but for her protection as the French king's beloved sister, verged on heresy. She or her circle may have encouraged Anne's interest in reform, as well as in poetry and literature. Anne's education in France proved itself in later years, inspiring many new trends among the ladies and courtiers of England, and it may have been instrumental in pressing their King toward the culture-shattering contretemps with the Papacy itself. Eric Ives's latest version of his biography hypothesizes that Anne may have had evangelist conviction and a strong spiritual inner life. William Forrest, author of a contemporary poem about Catherine of Aragon, complimented Anne's "passing excellent" skill as a dancer. "Here", he wrote, "was [a] fresh young damsel, that could trip and go."

Anne exerted a powerful charm on those who met her, though opinions differed on her attractiveness. The Venetian diarist Marino Sanuto, who saw Anne when Henry VIII met Francis I at Calais in October 1532, described her as "not one of the handsomest women in the world; she is of middling stature, swarthy complexion, long neck, wide mouth, bosom not much raised ... eyes, which are black and beautiful". Simon Grynée wrote to Martin Bucer in September 1531 that Anne was "young, good-looking, of a rather dark complexion". Lancelot de Carles called her "beautiful with an elegant figure", and a Venetian in Paris in 1528 also reported that she was said to be beautiful. The most influential description of Anne, but also the least reliable, was written by the Catholic propagandist and polemicist Nicholas Sanders in 1586, half a century after Anne's death: "Anne Boleyn was rather tall of stature, with black hair, and an oval face of a sallow complexion, as if troubled with jaundice. She had a projecting tooth under the upper lip, and on her right hand six fingers [which historians now know to be false]. There was a large wen under her chin, and therefore to hide its ugliness she wore a high dress covering her throat ... She was handsome to look at, with a pretty mouth". Sanders held Anne responsible for Henry VIII's rejection of the Catholic church, and writing fifty years after her death, was keen to demonize her. Sanders's description contributed to what biographer Eric Ives calls the "monster legend" of Anne Boleyn. Though his details were fictitious, they have formed the basis for references to Anne's appearance even in some modern textbooks.

Anne's experience in France made her a devout Christian in the new tradition of Renaissance humanism. While she would later hold the reformist position that the papacy was a corrupting influence on Christianity, her conservative tendencies could be seen in her devotion to the Virgin Mary. Anne's European education ended in 1521, when her father summoned her back to England. She sailed from Calais in January 1522.