Cornforth was educated at the university in his native city of Sydney and at Oxford University, where he obtained his DPhil in 1941. He spent the war in Oxford working on the structure of penicillin before joining the staff of the Medical Research Council in 1946. In 1962 Cornforth moved to the Shell research center at Sittingbourne in Kent to serve as director of the Milstead Laboratory of Chemical Enzymology. In 1975 he accepted the post of Royal Society Research Professor at Sussex University, where he served until 1982.

In 1951 the American chemist Robert Woodward had succeeded in synthesizing the important steroid, cholesterol; Cornforth was interested in how the molecule is actually synthesized in the cell. Using labeled isotopes of hydrogen, he traced out in considerable detail the chemical steps used to form the C₂₇H₄₆OH molecule of cholesterol from the initial CH₃COOH of acetic acid. It was for this work that he shared the 1975 Nobel Prize for chemistry with Vladimir Prelog. Cornforth has also synthesized alkenes, oxazoles, and the plant hormone abscisic acid.