In response, several Presbyterian ministers and congregations within the Canadian synod of the Church of Scotland switched their affiliation to the new denomination.

Named for Scottish Reformation theologian John Knox, the new college became affiliated with the Free Church.

Henry Esson on James Street, at the present site of Toronto Eaton Centre.

Dr. Michael Willis, the founding president of the Anti-Slavery Society of Canada (1851), became the first principal of the college in 1857.

Willis came to Toronto in 1846 from St. John's Renfield Church, Glasgow, where he followed Thomas Chalmers and took part in the Disruption of 1843.

Knox was formally granted its charter from the colonial government in 1858, thereby possessing the authority to confer academic degrees.

In 1875, Knox College moved to a new Gothic-revival building at 1 Spadina Crescent, and operated as the main seminary for the newly formed Presbyterian Church in Canada.

In 2005, Knox observed its 160th anniversary with a visit and lecture by Alison Elliot, the Moderator of the General Assembly of the Church of Scotland.

The pipework is modelled on the Johan Niclas Cahman organ at Leufsta Bruk, Sweden from 1726/28.

Notably, the Wolff organ is tuned to a modified fifth comma meantone temperament devised by Harald Vogel following 17th-century Swedish theorists.

In the Season 2, episode 12 of Star Trek: Discovery, "Through the Valley of Shadows", the college was used as a filming location for the Klingon monastery on Boreth.