Culp came to national attention with his first role on film as the lead in the 1957 western television series Trackdown; Steve McQueen's western TV show Wanted: Dead or Alive (1958-1961) was a spin-off of Culp's first series. Culp remains most famous for his role on the espionage series I Spy, opposite Bill Cosby, but in fact, he had had a film career after Trackdown and prior to that 1965 program, most notably in PT 109 and as Wild Bill Hickok in The Raiders (1963). He went on to star in the movie Bob & Carol & Ted & Alice (1969; probably the height of his career).

He played the murderer in three Columbo television movies, playing several different characters. In 1971, he, Peter Falk, Robert Wagner, and Darren McGavin all stepped in to take turns with Anthony Franciosa's rotation of The Name of the Game after Franciosa was fired, rotating the lead of the lavish 90-minute show about the magazine business with Gene Barry and Robert Stack. His next starring stint on television was as FBI agent Bill Maxwell in The Greatest American Hero (1981).

When Larry Hagman entered into contract negotiations over his character of J. R. Ewing in Dallas, Culp was ready to step into the role with an explanation that his face had been rebuilt following an accident. Culp also played the U.S. President in Alan J. Pakula's The Pelican Brief (1991). One of his most recent roles was a recurring part on Everybody Loves Raymond as Warren, Ray's father-in-law. Altogether, Culp has made dozens of appearances in TV shows and movies, with 86 roles to his credit between 1957 and 2005.

Culp lent his voice to the digital character Doctor Breen, the prime antagonist in the 2004 computer game Half-Life 2.

From 1967 to 1970, he was married to Eurasian actress France Nuyen.