MacCulloch attended Hillcroft Preparatory School and Stowmarket Grammar school in Suffolk, and subsequently read History at Churchill College, Cambridge (BA 1972, MA 1976), where he was organ scholar. He took a Diploma in Archive Administration at Liverpool University in 1973, and then returned to Cambridge to complete a PhD in Tudor History under the supervision of Sir G. R. Elton (awarded 1977), combining this with a position as Junior Research Fellow at Churchill College. MacCulloch was elected a Fellow of the Society of Antiquaries of London (1978), a Fellow of the Royal Historical Society (1982), and a Fellow of the British Academy (2001). He is a Doctor of Divinity of the University of Oxford (2001) and in 2003 was awarded an honourary DLitt by the University of East Anglia. He co-edits the Journal of Ecclesiastical History.

He joined the Gay Christian Movement in 1976, serving twice on its committee and briefly as honorary secretary. From 1978 until 1990, MacCulloch was a tutor at Wesley College, Bristol, and taught Church History in the department of Theology at the University of Bristol. He interrupted his teaching to study for the Oxford Diploma in Theology (awarded 1987) at Ripon College Cuddesdon. In 1987 he was ordained deacon in the Church of England and from 1987 to 1988 he served as Non-Stipendiary Minister at Clifton All Saints with St John in the Diocese of Bristol. However, in response to a motion put before the General Synod in 1987 by the Revd Tony Higton regarding the sexuality of clergy, he declined ordination to the priesthood and ceased to minister at Clifton.

Regarding the clash between his sexuality and the Church and his own retreat from religious orthodoxy he said:

I was ordained Deacon. But, being a gay man, it was just impossible to proceed further, within the conditions of the Anglican set-up, because I was determined that I would make no bones about who I was; I was brought up to be truthful, and truth has always mattered to me. The Church couldn't cope and so we parted company. It was a miserable experience.

His book Europe's House Divided 1490—1700 (2003) won the 2004 National Book Critics Circle Award and 2004 British Academy Book Prize, adding to his earlier success in carrying off the 1996 James Tait Black Memorial Prize for Thomas Cranmer: A Life. A History of Christianity: The First Three Thousand Years, was published in September 2009 with a related 6 part television series on BBC 4 in 2009 and BBC 2 and BBC 4 in 2010.