Ronald Theodore Kirk was born on August 7, 1935, in Columbus, Ohio, and grew up in the neighborhood known as Flytown. He lost his sight at about the age of two, which he attributed to improper medical treatment. He studied at the Ohio State School for the Blind, began on bugle and trumpet, then moved to clarinet and C-melody saxophone, and by fifteen was traveling on weekends with Boyd Moore's rhythm and blues band. The saxophonist Hank Crawford remembered the blind teenager playing two horns at once and tearing the joint up. As a teenager Kirk also found the manzello (a modified saxello with a larger, upturned bell) and the stritch (a straight alto saxophone with no upturned bell), and he began altering both, along with his tenor, so that he could hold several in his mouth at the same time. A dream led him to swap two letters of his first name and become Roland.

His first record, Triple Threat, was cut for King in New York on November 9, 1956, with a quartet led from the tenor, and appeared the following year. After working in Louisville he recorded Introducing Roland Kirk for Argo in Chicago on June 7, 1960, with Ira Sullivan on trumpet and tenor. In 1961 he made Kirk's Work for Prestige with organist Jack McDuff (July 11, at Van Gelder Studio) and the Mercury quartet sessions of August 16 and 17 that became We Free Kings, with Richard Wyands or Hank Jones at the piano and drummer Charlie Persip. That autumn he joined Charles Mingus's Jazz Workshop: a Birdland broadcast on October 21, 1961 caught him in the band, and on November 6 he played on the Atlantic session that produced Oh Yeah, released in April 1962, on which he doubled on flute, siren, tenor, manzello and stritch.

The years 1962 and 1963 were his busiest run on Mercury. He recorded most of Domino in New York on April 18, 1962 with Wynton Kelly at the piano and Roy Haynes on drums, and the rest in Chicago on September 6 with Andrew Hill; an April 17 session with Herbie Hancock at the piano was left in the vault and appeared on later reissues. In May 1962 he was the front line on Haynes's Out of the Afternoon for Impulse!, with Tommy Flanagan and Henry Grimes. That September he was the flute soloist on Quincy Jones's "Soul Bossa Nova," which later became familiar from the Austin Powers films. Reeds & Deeds (February 1963) and a June 1963 date with an orchestra arranged by Benny Golson followed. In October 1963 he toured Europe, recording the live album Kirk in Copenhagen at the Club Montmartre with pianist Tete Montoliu and the young bassist Niels-Henning Ørsted Pedersen, and making his first visit to London, where he played a season at Ronnie Scott's club.

Gifts & Messages (Los Angeles, July 1964) was followed by I Talk with the Spirits (Limelight, September 1964), a flutes-only album that contains the first recording of "Serenade to a Cuckoo." On January 13, 1965 he recorded Rip, Rig and Panic at Van Gelder Studio with Jaki Byard, bassist Richard Davis and Elvin Jones. A live set taped in New York that March, with Lonnie Liston Smith and Byard sharing the piano chair, was his first release for Atlantic, issued in 1967 as Here Comes the Whistleman. His Atlantic albums continued with The Inflated Tear (Webster Hall, November 1967), and the label released Left & Right with strings and the live and studio mix Volunteered Slavery, built from his July 7, 1968 Newport Jazz Festival set and July 1969 sessions. Ian Anderson of Jethro Tull, who recorded "Serenade to a Cuckoo" on the band's 1968 debut, met Kirk when both played the 1969 Newport festival.

By then Kirk had become a public figure beyond the clubs. With Mark Davis he founded the Jazz and People's Movement, which demanded more airtime and more work for Black jazz musicians. Its members disrupted The Merv Griffin Show in August 1970 with whistles and placards and broke into a taping of The Dick Cavett Show, which ended with producers agreeing to book jazz players the following week, among them Freddie Hubbard, Cecil Taylor and Andrew Cyrille. In January 1971 he was invited to The Ed Sullivan Show and brought a nine-piece band that included Mingus, Archie Shepp and Haynes, playing his own "The Inflated Tear" and Mingus's "Haitian Fight Song" instead of the pop tune the producers had requested. In 1970 he added Rahsaan to his name after hearing it in a dream, and the mayor of Columbus declared December 10, 1970 "Rahsaan Day." The records of that period include the live Rahsaan Rahsaan (Village Vanguard, May 1970), the spare Natural Black Inventions: Root Strata (early 1971), the soul and pop-oriented Blacknuss (1971), A Meeting of the Times with the singer Al Hibbler (March 1972), and Prepare Thyself to Deal with a Miracle (January 1973), which added strings and the singers Dee Dee Bridgewater and Jeanne Lee. In 1972 he played Montreux with pianist Les McCann and joined the all-star jam sessions of Newport in New York at Radio City Music Hall, and in June 1973 he recorded the double album Bright Moments at the Keystone Korner in San Francisco. He was a guest on Mingus's Carnegie Hall concert of January 19, 1974.

The Case of the 3 Sided Dream in Audio Color (1975) mixed tape loops, spoken commentary and a studio band, and also hid a telephone-message track on its apparently blank fourth side. In 1975 Kirk suffered a major stroke that left one side of his body partially paralyzed. He adapted his instruments so he could play with one hand, kept touring and recording, and made three albums for Warner Bros., The Return of the 5000 Lb. Man (1976), Kirkatron (1977) and Boogie-Woogie String Along for Real, along with the Atlantic release Other Folks' Music (1976). At a London engagement at Ronnie Scott's club after the stroke he still managed to play two instruments at once. A second stroke ended his life on December 5, 1977, the morning after a performance in the Frangipani Room of the Indiana University Student Union in Bloomington, Indiana. He was 42. His widow, Dorthaan Kirk, joined the public jazz station WBGO in Newark in 1978 and received the 2020 A.B. Spellman NEA Jazz Masters Fellowship for Jazz Advocacy. John Kruth's biography Bright Moments: The Life and Legacy of Rahsaan Roland Kirk appeared in 2000.

Kirk is remembered for the sight of three horns hanging from his neck, but his reputation among players rests on the music he made with them: he fingered the tenor with one hand and the manzello with the other while sounding a drone on the stritch, he held notes for long stretches with circular breathing, and he sang or hummed into the flute and paired a standard flute with a nose flute. The critic Gary Giddins wrote that his tenor playing alone would have secured his place, and called him the first major jazz innovator on flute after Eric Dolphy. He drew on the whole history of the music, from ragtime and the stride playing of Fats Waller and the clarinet of Sidney Bechet to John Coltrane and free playing, and he borrowed freely from classical composers and from pop writers such as Burt Bacharach and Smokey Robinson. He preferred the term "black classical music" for what he did, spoke to audiences between tunes about African-American history and politics, and brought alarm clocks, sirens and a length of garden hose he called the black mystery pipes into the performances. From the early 1970s his studio albums used tape manipulation and found sound. His work traveled well beyond jazz: Jethro Tull covered "Serenade to a Cuckoo," the British band Rip Rig + Panic took its name from his 1965 album, Frank Zappa named him in the liner notes of Freak Out!, the guitarist Charlie Hunter formed the band T. J. Kirk in tribute to Kirk, Monk and James Brown, and Pitchfork ranked The Inflated Tear number 170 on its 2017 list of the best albums of the 1960s.