Horne was born in Bradford, Pennsylvania. Her parents moved to Los Angeles when she was 11. She studied voice at the University of Southern California and participated in Lotte Lehmann's vocal classes.
Horne's first professional engagement was in 1954, when she dubbed the voice of Dorothy Dandridge in the film Carmen Jones. She made her stage Los Angeles debut the same year when she performed the role of Hata in The Bartered Bride with the Los Angeles Guild Opera.
Her first major breakthrough came when her singing ability was recognized by Igor Stravinsky; her operatic career began when he invited her to perform in the 1956 Vienna festival. She remained in Europe for three seasons singing for the Gelsenkirchen Opera. She was highly acclaimed for her performance as Marie in Berg's Wozzeck at the inauguration of Gelsenkirchen's new opera house on May 22, 1960
In 1964, she returned to the United States to appear in Wozzeck at the San Francisco Opera.
For many years, Horne was associated with the Australian soprano Joan Sutherland. They first performed together in a concert version of Bellini's Beatrice di Tenda at Carnegie Hall in February, 1961. In 1965, they were paired again in a performance of Rossini's Semiramide in Boston.
Horne made her debut at the Royal Opera House, Covent Garden in October 1964 as Marie in Wozzeck. Her La Scala debut was as Jocasta in Stravinsky's Oedipus Rex on March 13, 1969. Horne made her debut at the Metropolitan Opera in 1970 as Adalgisa in Bellini's Norma with Joan Sutherland in the title role. She thereafter appeared reguarly at the Met, opening the 1972-1973 season as Carmen. In 1984, she sang the title role of Handel's Rinaldo, the first Handel opera ever performed at the Met.
Horne was married many years to the conductor Henry Lewis.
Horne retired from the concert stage in 1999 with a recital at the Chicago Symphony Center.
Critics consider Horne the prototypical American singer; brisk, efficient, earnest, technically secure, but perhaps missing a touch of the irony and darkness of spirit of old world singers.