A graduate of Worcester Academy in 1922, Starrett went on to study at Dartmouth College. While on the Dartmouth football team he was hired to play a football extra in the film The Quarterback (1926). Bitten by the acting bug, Starrett played minor roles in films and leading roles in stage plays. In 1928, he was a member of the Walker Company, a repertory theatre troupe headed by Stuart Walker.

He played the romantic lead in Fast and Loose (1930), which also featured Miriam Hopkins, Carole Lombard, and Frank Morgan. He also starred in the Canadian production The Viking (1931), filmed on location in Newfoundland, which had begun as a Paramount Pictures project.

After that he was very active for the next two years but his roles were unremarkable. He was featured in Our Betters (1933), Murder on the Campus (1933). and in his most charming role as a young Doctor named Orion in "Along Came Love", with the vivacious co-star Irene Hervey. Offscreen, he helped organize the Screen Actors Guild.

Starrett's first western film was Gallant Defender (1935).

Charles Starrett retired at age 48, when his last Columbia contract lapsed. As Starrett had once taken over Columbia's westerns from Tim McCoy, Jock Mahoney took over the reins from Starrett, co-starring with Smiley Burnette in a new series. The pilot feature was completed but never released, so Columbia's long history of B westerns ended with Charles Starrett. Columbia serviced the still-strong demand for Starrett by reissuing his 1937-1940 westerns with the Sons of the Pioneers. These proved just as popular as the Durango series, and Columbia kept dozens of the Starrett features in theaters for several years.

After playing assorted rancher, ranger, and sheriff roles, Starrett was cast as "The Durango Kid" in 1940. The character was an upright citizen known and liked by the townsfolk, but he masqueraded as a notorious, black-garbed horseman to terrorize the local Criminals and foil their plans. The film was successful but not much different from some of Starrett's earlier good guy-chasing-bad-guy roles.

When Starrett's new contract lapsed in 1941, he withdrew from westerns and Columbia disbanded the unit. The Sons of the Pioneers moved to Republic Pictures, where they reunited with their former lead singer Roy Rogers.

Dub Taylor, as comic sidekick "Cannonball", worked with Starrett until 1946. At that time, Smiley Burnette, who had been a very popular sidekick to Gene Autry, was brought in to replace Taylor. Burnette, appropriately enough, played a character called "Smiley Burnette." The Durango Kid films combined vigorous action sequences—often with spectacular Stunts performed by Ted Mapes and later Jock Mahoney—and western music. Each film featured musical specialties by Burnette, and by a guest Artist or group from records or radio.

By 1949, the series faced a challenge. Production costs kept rising, but the financial return was limited. So Columbia resorted to shooting less new material, and borrowing scenes from older Durango Kid pictures to coax the running time up to the usual length. Columbia was starting to use this same recycling gambit in its adventure serials and Three Stooges comedies. Sometimes the scripting and editing were very clever, most memorably in Cyclone Fury (1951), in which footage from four older Starrett westerns is worked into the plot. The final Durango Kid feature was The Kid from Broken Gun (1952), with the new footage set in a courtroom and the old footage illustrating the testimony of the various characters.

Starrett hadn't planned on making an entire career out of westerns, and agreed to make them for two years, with the understanding that his bosses would then cast him in plainclothes roles. When they didn't, he walked out on his contract after the two years. (In 1974 Starrett told author James Horwitz that his self-imposed leave of absence cost him $60,000, which was the dollar value of his original agreement.) Meanwhile, theater exhibitors around the world were attracting big crowds with Charles Starrett westerns, so Columbia gave him a new contract with the actor insisting on appearing in a non-western. He finally got his chance—once—in 1937, for the collegiate musical comedy Start Cheering (released 1938). In a curious reflection of his own situation, Starrett played a disenchanted movie hero who wanted to do something different with his life. But Starrett's success in westerns established him firmly in outdoor fare and sealed his fate professionally. For the rest of his career he made Columbia westerns exclusively.

His fans never forgot him, and corresponded with him in later years. Starrett was pleased by the interest and made guest appearances at a few film conventions and revivals. Starrett died of cancer in Borrego Springs, California, in 1986, six days short of his 83rd birthday.

The musical westerns of Gene Autry inspired every Hollywood studio to have its Cowboy personalities use their musical talents—but not Charles Starrett. He could carry a tune but left the songs to professional vocalists (his vocals in Start Cheering were dubbed by Robert Paige). Columbia solved the Problem by hiring an entire singing group to support Starrett: the Sons of the Pioneers.