After attending Dulwich College, Guthrie went up to Cambridge University in 1925 winning the Eric Evan Spicer scholarship to Trinity College. He excelled in his studies, being supervised by, amongst others, Francis Cornford and A. S. F. Gow, and was placed in the first class of both Parts of the Classical Tripos, with distinction in Part II and the award of the Craven Prize.
After graduating he embarked on a postgraduate career at Trinity. He met his future wife, Adele Marion Ogilvie, an Australian studying at Newnham College, while supervising her undergraduate studies in 1929-1930. They married in 1933 and went on to have two children.
During the war he exchanged scholarship for military service serving in the Intelligence Corps between 1941 - 1945, based initially in London, then in St Albans and, from 1943, in Istanbul, achieving the temporary rank of Major.
In 1946 he was promoted to Reader before becoming the third Laurence Professor of Ancient Philosophy in 1952, the year in which he became a Fellow of the British Academy. In 1950 he edited an edition of his mentor Cornford's essays under the title The Unwritten Philosophy.
In 1957 he moved to his third Cambridge college when invited to become the Master of Downing College where he would remain for the rest of his life. As Master he took a full part in the administrative, cultural and social life of the college, occasionally preaching in the college chapel and supporting the undergraduate music club and boat club. He oversaw a rewriting of the college statutes and introduced a maximum term for a Master of fifteen years, by which he chose voluntarily to abide although it did not apply to him.
In 1956 he was approached by the Syndics of the Cambridge University Press to write a history of ancient philosophy. The first volumes, devoted to the pre-Socratics, of what would be his life's magnum opus were published to high acclaim in 1962 and 1964. The work continued while serving as Master of Downing and became his life's full mission after retiring from that position in 1972. The venture remained, however, unfinished at his death aged 74 in 1981 the year in which he published the sixth volume in the series, devoted to Aristotle.
As a philosopher Guthrie followed in the tradition of Cornford in believing that ancient philosophers should be read and interpreted against their own historical background, rather than engaged with, as has been the practice of later generations of classical philosophers, in the context of the whole canon of philosophy both ancient and modern.