MacCulloch attended Hillcroft Preparatory School and Stowmarket Grammar school in Suffolk, and subsequently read History at Churchill College, Cambridge (B.A. 1972, M.A. 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 Ph.D. 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 D.Litt. by the University of East Anglia. He co-edits the Journal of Ecclesiastical History.

He joined the (now Lesbian and) 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 Saint John in the diocese of Bristol. However, in response to a motion put before the General Synod in 1987 by the Reverend Tony Higton regarding sexuality of clergy, he declined ordination to the priesthood and ceased to minister at Clifton.

His book Reformation: Europe's House Divided 1490–1700 (2003) won the 2004 National Book Critics Circle Award adding to his earlier success in carrying off the 1996 James Tait Black Memorial Prize for Thomas Cranmer: A Life. His latest book, Christianity: the first 3000 years, will be published in 2009, to accompany a Television series on BBC 4.