John Macgregor was born on 3 October 1946, the son of Dr D. F. Macgregor and K. A. Macgregor (née Adams). He was educated at Kibworth Beauchamp Grammar School, Leicester and Balliol College, University of Oxford (taking a BA in Music, 1967; MA 2010), also taking a Certificate of Education at Birmingham University in 1969. In 1982, he married Judith Anne Brown, having three sons and one daughter.

After teaching music at Cranleigh School, Surrey, 1969-73, Macgregor joined the Diplomatic Service in 1973, and his diplomatic career included: 2nd Secretary, United Nations Affairs, 1973-74; 1st Secretary (political), New Delhi [India], 1975; Foreign and Commonwealth Office, 1979; Private Secretary to Minister of State, FCO, 1981; speechwriter to Foreign Secretary, 1982; Assistant, Soviet Department, FCO, 1983; Deputy Head of Mission, Prague [Czechoslovakia, now Czech Republic and Slovakia], 1986; Head of Chancery, Paris [France], 1990-93; Head of EU Department (External), 1993-95; Director General for Trade Promotion in Germany and Consul-General, Düsseldorf, 1995-98; Ambassador to Poland, 1998-2000; Director, Wider Europe, FCO, 2000-02; Ambassador to Austria, 2003-07; UK Permanent Representative to UN in Vienna and UK Government to IAEA, 2006-07.