He taught physiology and biology in Saint Louis, Missouri and Puerto Rico, and he embarked on natural history expeditions to the Orinoco River in South America and Cuba.

In 1912 he was appointed a research associate at the Carnegie Institution's Cold Spring Harbor Laboratory in Long Island.

[2] His research spanned endocrinology, the physiology of reproduction, animal pigmentation, and the nature and functional basis of sex.

[2][5] Also around 1932 Riddle was earliest to show that the prolactin protein induces the secretion of crop milk in pigeons and other birds.

[2] Riddle was a devout atheist and held the conviction that religion poses a serious threat to scientific advancements.