By Papal Bull dated 14 October 1504, Fisher was appointed the Bishop of Rochester at the personal insistence of Henry VII. Rochester was then the poorest diocese in England and usually seen as a first step on an ecclesiastical career. Nonetheless, Fisher stayed there, presumably by his own choice, for the remaining 31 years of his life. At the same time, like any English bishop of his day, Fisher had certain state duties. In particular, he maintained a passionate interest in the University of Cambridge. In 1504 he was elected the university's chancellor. Re-elected annually for 10 years, Fisher ultimately received a lifetime appointment. At this date he is also said to have acted as tutor to Prince Henry, afterwards King Henry VIII. As a preacher his reputation was so great that Fisher was appointed to preach the funeral oration for King Henry VII and the Lady Margaret, both of whom died in 1509, the texts being extant. Besides his share in the Lady Margaret's foundations, Fisher gave further proof of his zeal for learning by inducing Erasmus to visit Cambridge. The latter attributes it ("Epistulae" 6:2) to Fisher's protection that the study of Greek was allowed to proceed at Cambridge without the active molestation that it encountered at Oxford.

In 1512 Fisher was nominated as one of the English representatives at the Fifth Council of the Lateran, then sitting, but his journey to Rome was postponed, and finally abandoned.

Fisher has also been named, though without any real proof, as the true author of the royal treatise against Martin Luther entitled "Assertio septem sacramentorum" (Defence of the Seven Sacraments), published in 1521, which won for King Henry VIII the title "Fidei Defensor" (Defender of the Faith). Prior to this date Fisher had denounced various abuses in the church, urging the need for disciplinary reforms. On about 11 February 1526, at the King's command, he preached a famous sermon against Luther at St Paul's Cross, the open-air pulpit outside St Paul's Cathedral in London. This was in the wake of numerous other controversial writings; the battle against heterodox teachings increasingly occupied Fisher's later years. In 1529 Fisher ordered the arrest of Thomas Hitton, a follower of william Tyndale, and subsequently interrogated him. Hitton was tortured and executed at the stake for heresy.

In November 1529, the "Long Parliament" of Henry's reign began encroaching on the Catholic Church's prerogatives. Fisher, as a member of the upper house, the House of Lords, at once warned Parliament that such acts could only end in the utter destruction of the Catholic Church in England. The Commons, through their speaker, complained to the King that Fisher had disparaged Parliament, presumably with Henry prompting them behind the scenes. The opportunity was not lost. Henry summoned Fisher before him, demanding an explanation. This being given, Henry declared himself satisfied, leaving it to the Commons to declare that the explanation was inadequate, so that he appeared as a magnanimous sovereign, instead of Fisher's enemy.

A year later, in 1530, the continued encroachments on the Church moved Fisher, as Bishop of Rochester, along with the Bishops of Bath and Ely, to appeal to the Holy See. This gave the King his opportunity and an edict forbidding such appeals was immediately issued, and the three bishops were arrested. Their imprisonment, however, must have lasted only a few months for in February 1531, Convocation met, and Fisher was present. This was the occasion when the clergy were forced, at a cost of 100,000 pounds, to purchase the King's pardon for having recognized Cardinal Wolsey's authority as legate of the pope; and at the same time to acknowledge Henry as Supreme Head of the Church in England, to which phrase the addition of the clause "so far as God's law permits" was made through Fisher's efforts.

Fisher also engaged in secret activities to overthrow Henry. As early as 1531 he began secretly communicating with foreign diplomats. In September 1533 communicating secretly through the imperial ambassador Eustace Chapuys he encouraged Holy Roman Emperor Charles V to invade England and depose Henry in combination with a domestic uprising.

Matters now moved rapidly. In May 1532, Sir Thomas More resigned the chancellorship and, in June, Fisher preached publicly against the divorce. In August, william Warham, Archbishop of Canterbury, died and Thomas Cranmer was at once proposed by Henry to the Pope as his successor. In January of the next year, Henry secretly went through a form of marriage with Anne Boleyn. Cranmer's consecration as a bishop took place in March 1533, and, a week later, Fisher was arrested. It seems that the purpose of this arrest was to prevent him from opposing the sentence of divorce which Cranmer pronounced in May, or the coronation of Anne Boleyn which followed on 1 June, for Fisher was set at liberty again within a fortnight of the latter event, no charge being made against him. In the autumn of 1533, various arrests were made in connection with the so-called revelations of the Holy Maid of Kent, Elizabeth Barton, but as Fisher was taken seriously ill in December, proceedings against him were postponed for a time. However, in March 1534, a special Bill of Attainder against Fisher and others for complicity in the matter of the Maid of Kent was introduced in Parliament and passed. By this, Fisher was condemned to forfeit all his personal estate and to be imprisoned during the King's pleasure. Subsequently a pardon was granted him on payment of a fine of 300 pounds.

The same session of Parliament passed the First Succession Act, by which all who should be called upon to do so were compelled to take an oath of succession, acknowledging the issue of Henry and Anne as legitimate heirs to the throne, under pain of being guilty of misprision of treason. Fisher refused the oath and was imprisoned in the Tower of London on 26 April 1534. Several efforts were made to induce him to submit, but without effect, and in November he was attained of misprision of treason a second time, his goods being forfeited as from the previous 1 March, and the See of Rochester being declared vacant as of 2 June following. He was to remain in the Tower for over a year, and while he was allowed food and drink sent by friends, and a servant, he was not allowed a priest, even to the very end. A long letter exists, written from the Tower by Fisher to Thomas Cromwell, speaking of the severity of his conditions of imprisonment.

However, a public outcry was brewing among the London populace who saw a sinister irony in the parallels between the conviction of Fisher and that of his patronal namesake, Saint John the Baptist, who was executed by King Herod Antipas for challenging the validity of Herod's marriage to his brother's divorcée Herodias. For fear of John Fisher's living through his patronal feast day, that of the Nativity of St. John the Baptist on 24 June, and of attracting too much public sympathy, King Henry commuted the sentence to that of beheading, to be accomplished before 23 June, the Vigil of the feast of the Nativity of St. John the Baptist. He was executed on Tower Hill on 22 June 1535. The execution had the opposite effect from that which King Henry VIII intended, as it created yet another parallel with that of the martyrdom of St. John the Baptist, who was also beheaded; his death also happened on the feast day of Saint Alban, the first martyr of Britain.

Fisher was beatified by Pope Leo XIII with Thomas More and 52 other English Martyrs on 29 December 1886 and canonised, with Thomas More, on 19 May 1935 by Pope Pius XI. His feast day, for celebration jointly with St Thomas More, is on 22 June (the date of Fisher's execution). In 1980, despite being an opponent of the English Reformation, Fisher was added to the Church of England's calendar of Saints and Heroes of the Christian Church, jointly with Thomas More, to be commemorated every 6 July (the date of More's execution) as "Thomas More, Scholar, and John Fisher, Bishop of Rochester, Reformation Martyrs, 1535".

John Fisher was portrayed by veteran actor Joseph O'Conor in the film Anne of the Thousand Days (1969), by Bosco Hogan in the miniseries The Tudors, and by Geoffrey Lewis in the 1971 miniseries The Six Wives of Henry VIII.

A list of Fisher's writings is found in Joseph Gillow's Bibliographical Dictionary of the English Catholics (London, s.d.), II, 262–270. There are twenty-six works in all, printed and manuscript, mostly ascetical or controversial treatises, several of which have been reprinted many times. The original editions are very rare and valuable. The principal are: