Du Vigneaud studied at the University of Illinois, Urbana, took his doctorate from the University of Rochester, N.Y. (1927), then studied at Johns Hopkins University, Baltimore, the Kaiser Wilhelm Institute, Berlin, and the University of Edinburgh. He headed the biochemistry department of the George Washington University Medical School, Washington, D.C. (1932–38), and was professor and head of the department of biochemistry at the Cornell University Medical College, New York City (1938–67), and professor of chemistry at Cornell University, Ithaca, N.Y. (1967–75).

Du Vigneaud and his staff at Cornell helped identify the chemical structure of the hormone insulin in the late 1930s, and in the early 1940s they established the structure of the sulfur-bearing vitamin biotin. Later that decade, they isolated vasopressin and oxytocin and analyzed both those hormones' chemical structure. Du Vigneaud found that the oxytocin molecule contains only eight different amino acids, in contrast to the hundreds of amino acids most other proteins contain. In 1953 he was able to synthesize oxytocin, becoming the first to achieve the synthesis of a protein hormone. In 1946 du Vigneaud and his colleagues at Cornell achieved another breakthrough, the synthesis of penicillin.