His guitar-playing father introduced him to the music of Blind Lemon Jefferson, whose music became a major influence. During the early 1940s Lenoir worked with blues artists, Sonny Boy Williamson, and Elmore James in New Orleans, Louisiana, and also became influenced by Arthur Crudup and Lightnin' Hopkins.
In 1949, he moved to Chicago, and Big Bill Broonzy helped introduce him to the local blues community. He began to perform at local clubs with fellow musicians including Memphis Minnie, Big Maceo Merriweather, and Muddy Waters, and became an important part of the city's blues scene. He first recorded in late 1950 for the J.O.B. label, and his recording of "Korea Blues" was licensed to and released by Chess as by "J. B. and his Bayou Boys". His band included pianist Sunnyland Slim, guitarist Leroy Foster, and drummer Alfred Wallace.
During the early 1950s Lenoir recorded on various labels in the Chicago area including J.O.B., Chess, Parrot, and Checker. His more successful songs included "Let's Roll", "The Mojo" featuring saxophonist J. T. Brown, and the controversial "Eisenhower Blues" which his record company, Parrot, forced him to re-record as "Tax Paying Blues".
Lenoir was known in the 1950s for his showmanship - in particular his zebra-patterned costumes - and his high-pitched vocals. He became a very influential electric guitarist and songwriter, and his penchant for social commentary distinguished him from many other bluesmen of the time. His most commercially successful and enduring release was "Mamma Talk To Your Daughter", recorded for Parrot in 1954, which reached # 11 on the Billboard R&B chart and was later recorded by many other blues and rock musicians. Lenoir's sound was unique: saxes (usually Alex Atkins and Ernest Cotton) wailed in unison behind Lenoir's boogie-driven rhythm guitar as drummer Al Galvin pounded out a rudimentary backbeat. In the later 1950s, recording on the Checker label, he wrote several more blues standards including "Don't Dog Your Woman" and "Don't Touch My Head".
By 1960 he had moved to Vee Jay Records, and in 1963 he recorded for USA Records as "J. B. Lenoir and his African Hunch Rhythm", developing an interest in African percussion. However, he struggled to work as a professional musician and for a time took menial jobs, including working in the kitchen at the University of Illinois in Champaign. While there, he was rediscovered by Willie Dixon, who recorded him with drummer Fred Below on the albums Alabama Blues (1965) and Down In Mississippi (1966), inspired by the Civil Rights and Free Speech movements. The albums were first released in Germany by blues promoter Horst Lippmann. Lenoir toured Europe, and performed in 1965 with the American Folk Blues Festival in England.
Despite the angry lyrics of many of his songs, Lenoir sang in a disarmingly sweet, laid-back style, and he was widely known as an exceptionally friendly and gentle person. He befriended and encouraged many young blues artists both black and white.
The 2003 documentary film The Soul of a Man, directed by Wim Wenders as the fourth instalment of Martin Scorsese's series "The Blues", explored Lenoir's career, together with those of Skip James and Blind Willie Johnson.