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Turturro was born in Brooklyn, New York, the son of Katherine, an amateur jazz singer who worked in a Navy yard during World War II, and Nicholas Turturro, a carpenter and construction worker who immigrated from Giovinazzo, Italy at the age of six and fought as a Navy serviceman in D-Day. He was raised a Catholic and moved to the Rosedale section of Queens, New York with his family when he was six. He majored in drama at the State University of New York at New Paltz, and completed his MFA at the Yale School of Drama. He first appeared on film working as an extra in Martin Scorsese's critically acclaimed Raging Bull (1980).

Turturro created the title role of John Patrick Shanley's Danny and the Deep Blue Sea at the Playwrights Conference at the Eugene O'Neill Theatre Center in 1983. He repeated it the following year off-Broadway and won an Obie Award. Spike Lee liked Turturro's performance in Five Corners so much that he chose to cast him in Do the Right Thing. This movie was the first of a long-standing collaboration between the famous director and John Turturro, which also includes Mo' Better Blues (1990), Jungle Fever (1991), Clockers (1995), Girl 6 (1996), He Got Game (1998), Summer of Sam (1999) and She Hate Me (2004).

A versatile actor comfortable with both comedy and drama, Turturro also had an extended collaboration with the Coen Brothers, appearing in their films Miller's Crossing (1990), Barton Fink (1991), The Big Lebowski (1998), and O Brother, Where Art Thou? (2000). He has also appeared in several of Adam Sandler's movies, such as Mr. Deeds (2002) and You Don't Mess with the Zohan (2008). He played a severely disturbed patient of Jack Nicholson's in the comedy Anger Management and played Johnny Depp's antagonist in Secret Window. Turturro is also an occasional guest star on Monk as Adrian's eccentric brother, Ambrose Monk. Before becoming a household name, Turturro made a cameo in the Woody Allen film Hannah and Her Sisters. One of his comedy performances has attracted a cult following: his breezy take on Groucho Marx in the neglected 1992 comedy Brain Donors, an update of A Night at the Opera starring Turturro as an ambulance-chasing lawyer.

He won an Emmy award for his portrayal of Adrian Monk's brother Ambrose Monk in the USA Network series Monk. He has also been nominated and won many awards from many film organizations such as Screen Actors Guild, Cannes Film Festival, Golden Globes, and others.[4]

Turturro produced and directed, as well as acted in, the film Illuminata (1999), which also starred his wife Katherine Borowitz. He also wrote and directed the film Romance and Cigarettes (2005). He recently appeared in Robert De Niro's The Good Shepherd (film) as the right hand man of C.I.A. man Edward Wilson (Matt Damon), and as the oddball Sector 7 agent Simmons in Michael Bay's Transformers. He reprises the role in the sequel, Revenge of the Fallen.

John's brothers are actor Nicholas Turturro, and middle school art teacher, Ralph Turturro. Actress Aida Turturro is John Turturro's cousin. He has two children, Amedeo and Diego Turturro. Turturro is Roman Catholic and his wife, actress Katherine Borowitz, is Jewish.