Born in Atlanta, Georgia, Knight first launched to fame with The Pips in 1961, with "Every Beat of My Heart". In spite of a few more hits, Knight did not achieve widespread success until 1966, after signing to Motown. "Friendship Train", "I Heard It Through the Grapevine" and "If I Were Your Woman" became huge hits, though her biggest was "Neither One of Us". In the mid 1970s, the group hit its popular and critical peak with "Midnight Train to Georgia", "I've Got to Use My Imagination" and "Best Thing that Ever Happened to Me". She also joined with Dionne Warwick, Stevie Wonder, and Elton John on the 1986 AIDS benefit single, "That's What Friends Are For".
Gladys Knight has continued touring and recording occasionally, though the Pips have retired.
Gladys Knight was married to Barry Hankerson, uncle of singer Aaliyah.