Born in Chicago, Illinois to an Italian-American family, he made his acting debut in the 1969 stage production of Hair.

Mantegna has been married to the former Arlene Vrhel since December 3, 1975. They have two children of whom one - their daughter Mia - is autistic.

He debuted on Broadway in Working (1978). He also helped write Bleacher Bums, an award-winning play, which was first performed at Chicago's Organic Theater. He won a Tony award for his portrayal of Richard Roma in David Mamet's play Glengarry Glen Ross.

He made his feature motion picture debut in Medusa Challenger (1977). He played womanizing dentist Bruce Fleckstein in Compromising Positions (1985). Other early movies include co-starring roles in The Money Pit (1986), Weeds (1987) and Suspect (1987). He also starred in the critically acclaimed movies House of Games (1987) and Things Change (1988), for which he and co-star, the late Don Ameche, both received the Best Actor Award at the Venice Film Festival. In 1991 he starred in the highly praised police thriller Homicide.

Mantegna also has a recurring role in the animated series The Simpsons as mob boss Anthony "Fat Tony" D'Amico. He insists on voicing the character every time he appears, no matter how little dialogue he has. To quote: "If Fat Tony sneezes I want to be there". He received the Lifetime Achievement Award on April 26, 2004, at the Los Angeles Italian Film Festival.