In 1898 he received his medical degree from University of Aberdeen and went to work for a year at the University of Leipzig. In 1899 he was appointed Demonstrator of Physiology at the London Hospital Medical School and in 1902 he was appointed Lecturer in Biochemistry at the school. In 1903 he was appointed Professor of Physiology at the Western University at Cleveland, Ohio. In 1918 he was elected Professor of Physiology at the University of Toronto, Canada.
Macleod's main work was on carbohydrate metabolism and his efforts with Frederick Banting and Charles Best in the discovery of insulin. For this Banting and Macleod were jointly awarded the Nobel Prize for Medicine in 1923.
He wrote eleven books, including Recent Advances in Physiology (1905); Diabetes: its Pathological Physiology (1925); and Carbohydrate Metabolism and Insulin (1926)
There is a persistent rumour that he did not participate in the discovery of insulin. It is possible that he lent his lab facilities to Banting and Best and was away on a fishing trip when the discovery was actually made.