Born in Cincinnati, Ohio, Riddle was educated at Indiana University and at Chicago, where he obtained his PhD in 1907. After serving on the Chicago faculty from 1904 to 1911, he joined the research staff of the Carnegie Institution at their Station for Experimental Evolution, Cold Spring Harbor, New York, where he remained until his retirement in 1945.

In 1928 it was found that an extract from the anterior pituitary would stimulate milk secretion in rabbits. Riddle and his colleagues soon succeeded in isolating the hormone, which he named prolactin in 1932, and began a prolonged study of its physiological effects. He discovered that it would stimulate the growth of the crop sac in pigeons and inhibit gonadal growth in a number of animals.

His most dramatic and controversial finding, however, was that he could induce maternal behavior in hens by the injection of prolactin. Rats too were shown to adopt such normal maternal behavior as licking and retrieving despite their virgin state.