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.

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 current Knox College building, completed in 1915, was designed by architectural firm Chapman & McGiffin and reflects the Collegiate Gothic style that was once popular in North America.

The Gothic style of the structure is evident throughout, but is most accentuated inside the lobby in the eastern wing of the building (at the entrance from King's College Circle), which is characterized by columns rising into fan vaults.

It is an historically oriented organ in the North German baroque style, built as Opus 33 by Wolff & Associés of Laval, Quebec.

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.