In 1960, Redding began touring the South with Johnny Jenkins and The Pinetoppers. That same year he made his first recordings, "She's All Right" and "Shout Bamalana" with this group under the name "Otis and The Shooters".

In 1962, he made his first real mark in the music business during a Johnny Jenkins session when he recorded "These Arms of Mine," a ballad that Redding himself had written. The song became a minor hit on Volt Records, a subsidiary of renowned "Southern soul" label Stax, based in Memphis, Tennessee. His manager was fellow Maconite Phil Walden (who later founded Capricorn Records). Otis Redding continued to release for Stax/Volt, and built his fanbase by extensively touring a legendarily electrifying live show with support from fellow Stax artists Sam and Dave. Further hits between 1964 and 1966 included "Mr. Pitiful", "I Can't Turn You Loose", "Try a Little Tenderness", "(I Can't Get No) Satisfaction" (The Rolling Stones song), and "Respect" (later a smash hit for Aretha Franklin).

Redding wrote many of his own songs, which was unusual for the time, often with Steve Cropper (of Stax house band Booker T & the MG's, who usually served as Otis' backing band in the studio). Soul singer Jerry Butler co-wrote another hit "I've Been Loving You Too Long". One of his few songs with a significant mainstream following was "Tramp" (1967) with Carla Thomas. Later that year, Redding played at the massively influential Monterey Pop Festival.

Redding and six others were killed when the plane on which they were traveling crashed into Lake Monona in Madison, Wisconsin on December 10, 1967. Ben Cauley, one of the members of Redding's backup band, The Bar-Kays, was the only person aboard the plane to survive. He had been asleep until just seconds before impact, and recalled that upon waking he saw bandmate Phalon Jones look out a window and say, "Oh, no!" Cauley then unbuckled his seat belt, and that was his final recollection before finding himself in the frigid waters of the lake, grasping a seat cushion to keep himself afloat. The cause of the crash was never precisely determined.

Redding was laid to rest in a tomb on his private ranch in Round Oak, Georgia, about 20 miles north of Macon.

"(Sittin' on) the Dock of the Bay" was recorded only three days prior to Redding's death. It was released the next month and became his first #1 single and first million-seller. The fact that "(Sittin' on) the Dock of the Bay" ultimately became Redding's greatest commercial success is somewhat ironic, not only because its release came after his death, but also because the song is actually a significant stylistic departure from the bulk of his other work.

A few further records were posthumously released, including "Hard to Handle" (1968).

His sons Dexter and Otis III founded together with cousin Mark Locket the funk/disco-band "The Reddings" in the late 1970s.

In 2002, the city of Macon honored its native son, unveiling a memorial statue of Redding in the city's Gateway Park.