Thurber attended Ohio State University from 1913 to 1918 and left without taking a degree. He held several newspaper jobs before going in 1926 to New York City, where he was a reporter for the Evening Post. In 1927 he joined Harold Ross's newly established magazine, The New Yorker (New Yorker, The), as managing editor and staff writer, making a substantial contribution to setting its urbane tone. He was later to write an account of his associates there in The Years with Ross (1959).

His first published drawing in the magazine appeared in 1931. He considered himself primarily a writer and had been offhand about his sketches. But his friend, the essayist E.B. White (White, E.B.), noticed their worth and had them used as illustrations for their jointly written Is Sex Necessary? (1929), a spoof on the then-popular earnest, pseudoscientific approach to sex. Thurber's stock characters—the snarling wife, her timid, hapless husband, and a roster of serene, silently observing animals—have become classics of urban mythology.

After Thurber left The New Yorker staff in 1933, he remained a leading contributor. In 1940, failing eyesight, the result of a boyhood accident (he had lost use of his left eye at age 6), forced him to curtail his drawing, and by 1952 he had to give it up altogether as his blindness became nearly total.

My Life and Hard Times (1933) is a whimsical group of autobiographical pieces; a similar collection of family sketches appeared later in The Thurber Album (1952). His Fables for Our Time (1940) are deceptively simple and charming in style, yet unflinchingly clear-sighted in their appraisal of human foibles. A play, The Male Animal (1941), written with Elliott Nugent, is a plea for academic freedom as well as a comedy. His fantasies for children, The 13 Clocks (1950) and The Wonderful O (1957), are among the most successful fairy tales of modern times. The Thurber Carnival (1945), a collection of his writings and drawings, was adapted for the stage in 1960, with Thurber playing himself. A further collection, Credos and Curios, was published posthumously in 1962.