Born in Chicago, Illinois, Du Vigneaud graduated from the University of Illinois in 1923; he remained there to take his master's degree before going to the University of Rochester. There he studied the hormone insulin, gaining his PhD in 1927. The research on insulin marked the beginning of his interest in sulfur compounds, particularly the sulfur-containing amino acids – methionine, cystine, and cysteine.

In 1938 Du Vigneaud became head of the biochemistry department of Cornell University Medical College. Two years later he had isolated vitamin H (biotin) and by 1942 had determined its structure. He then went on to examine the hormones secreted by the posterior pituitary gland, especially oxytocin and vasopressin. He found oxytocin to be composed of eight amino acids, worked out the order of these, and in 1954 synthesized artificial oxytocin, which was shown to be as effective as the natural hormone in inducing labor and milk flow. This was the first protein to be synthesized and for this achievement Du Vigneaud received the Nobel Prize for chemistry in 1955.

Du Vigneaud's other work included research on penicillin and on methyl groups. He was professor of chemistry at Cornell University from 1967 to 1975 and subsequently emeritus professor of biochemistry there.