The Reformation and the religious changes of the mid-sixteenth century left their mark on the College. John Warner, the Regius Professor of Medicine (Warden from 1536 to 1556 and again from 1559 to 1565) showed notable flexibility in retaining office under Henry VIII, Edward VI, Mary I (for two years), and Elizabeth I, but some Fellows were deprived of their fellowships in both Protestant and Catholic times. The chapel lost its altars, its reredos statues, its organ, and some of its vestments and ornaments; changes in taste as well as religion caused many manuscripts to be withdrawn from the College library, some of which (thanks to the enterprise of Christopher Plantin) are now in the Plantin-Moretus Museum in Antwerp. Such restoration as occurred under Philip and Mary was reversed under their successor, when the chantry functions of the College became much attenuated and a clean sweep was ultimately made of 'idolatrous monuments'. The religious changes of the sixteenth century also affected the college's academic bias. Greek and Latin lecturerships and a Civil Law lecture (seemingly short lived) were established by the Royal Visitors in 1535, and the Commissioners of 1549 even proposed that the College should become devoted exclusively to legal studies. This did not come about (the twenty-four theology fellowships continued to be formally 'artist' places), but the College's reputation as a nursery of lawyers, and especially of civil lawyers practising in the ecclesiastical and admiralty courts, dates from this period.

The post-Reformation years were a time of indiscipline within the College. Archbishop Cranmer's visitation of 1541 had shown that non-residence, failure to take degrees and enter holy orders, sumptuary extravagance, and brawling and drunkenness were not unknown among the Fellows, and that – more seriously – the practice of corrupt resignation (the acceptance of payment for nomination of a successor to an arranged vacancy in the fellowship) was already rife. The College clearly needed a strong guiding hand; it found one in Warden Hovenden.