The College's second century brought the Reformation and difficult times. Lincoln was a small and relatively poor society, consisting of a Rector and Fellows, all in priests' orders, a few resident graduates and a sprinkling of junior members, most of whom were deeply attached to the traditions of the past and opposed to the changes made by Henry VIII's government. In 1527, Rector Cottisford had let the cellar under his lodgings be used as a prison for those suspected of propagating Lutheran ideas (one of the prisoners actually died of a sickness he had caught in the cellar). Cottisford and his colleagues, if reluctantly, concurred in the new church settlement. But his successor as Rector, Hugh Weston, was high in the favour of Catholic Queen Mary, who made him Dean of Westminster. He was chairman of the commissioners appointed in 1554 to dispute with the Protestant Archibishop Cranmer, and Bishops Latimer and Ridley before their martyrdom. (Latimer and Ridley were burned at the stake in Broad Street, Oxford.) The eleventh Rector, Henry Henshaw, and some of the Fellows, also loyal Catholics, were expelled by Queen Elizabeth I's Visitors.
Many in Lincoln remained attached to the old faith. Four Rectors were imposed on the College by Queen Elizabeth's favourite, Robert Dudley, Earl of Leicester, Chancellor of the University, in contravention of the statutes which required that the Rector should be chosen from Fellows or ex-Fellows; two of these, Francis Babington and John Bridgwater, turned out to be Romanists and had to escape into exile. The pupils of such men included some who suffered the ultimate penalty for their faith. William Filbie was executed at the Tower of London in 1582, praying for Queen Elizabeth and denying that the Pope was her enemy. William Hart was hanged, drawn and quartered at York in 1583. When he expired a number of those in the crowd that saw him die struggled 'who should first touch and seize for himself either coats or boots or any part of the martyr's clothes'. Another Romanist of Lincoln, William Gifford, went abroad and in 1621 became Archbishop of Rheims and Primate of France. It is unsurprising to note the documentary evidence that suggests the government found it necessary to install a spy in the College during these difficult decades.
In spite of its Catholic leanings, Lincoln eventually adapted to the new political and social imperatives of the late sixteenth and early seventeenth centuries. The College was closely associated with the production of the 'King James' or Authorised Version of the Bible in 1611, its 16th Rector Richard Kilby and another Lincoln man, Richard Brett, helping with the task of translation. At the same time a fundamental change in the character of the College was taking place. Undergraduate education had traditionally been carried out in residential halls rather than the colleges, but with the disappearance of the halls the colleges became much more important, especially as an increasing number of young men, many of them children of the gentry and aristocracy, sought entry at Oxford. Fellows ceased to be merely scholars and, in modern parlance, researchers, and became tutors of the junior members, exercising complete supervision over their finances, discipline and work.