Born to William West Toole Jr. and Dorothy Geraldine Niland, Annette began acting at a very young age, doing guest appearances in shows such as My Three Sons (1960), The Virginian (1962), Gunsmoke (1955), and The Partridge Family (1970).

Although she appeared in a number of television and theatrical movies from 1973 onward (notably as the ecdysiastical beauty pageant contestant in Michael Ritchie's Smile (1975) and as Tammy Wynette in 1981's Stand By Your Man), her first major role was as Nick Nolte's loyal girlfriend in 48 Hours, which came out in 1982. In the same year, she appeared opposite Nastassja Kinski in Cat People.

1983 saw her play the part of Lana Lang in Superman III opposite Christopher Reeve.

In 1985 she appeared in the television movie Copacabana.

In 1990 she starred in a remake of A Girl of the Limberlost and in Stephen King's It.

She had recurring roles on the television shows Nash Bridges (1996) and The Huntress (2000). She has also performed in a number of successful theater productions. In October 2001, she returned to the Superman mythos in the role of Martha Kent, Clark Kent/Superman's mother, in the television series Smallville, a role she continues to play today. She is thus the only actress ever to play both Superman's girlfriend and Superman's mother.

O'Toole has been married to actor Michael McKean since 1999, and in 2003 the duo were nominated for the Academy Award for Best Song for A Kiss at the End of the Rainbow, a love duet that they had written for the movie A Mighty Wind. O'Toole and McKean guest-starred together in an episode of Law & Order as a wealthy married couple accused of murder. McKean also became part of the Superman mythos when he guest starred on Smallville playing Perry White.