Born in Somerville, New Jersey, Van Cleef served in the United States Navy during World War II and became an actor after a brief career as an accountant. His first film was the classic Western High Noon, in which he played a villain. He also had a bit part as the sharpshooter in the climax of The Beast from 20,000 Fathoms around the same time.
Van Cleef played one of Lee Marvin's villainous henchmen in the 1962 John Ford classic The Man Who Shot Liberty Valance, with James Stewart and John Wayne.
He lost the tip of his middle finger on his right hand at some point: this can be seen in the close-up shots of his hand during the gunfights in The Good, the Bad, and the Ugly.
He appeared in several Spaghetti westerns, including in For a Few Dollars More and The Good, the Bad and the Ugly (both co-starring Clint Eastwood), as well as The Big Gundown and The Sabata Trilogy. Van Cleef also had a supporting role in John Carpenter's cult hit Escape from New York. He also appeared as a villainous swindler in the Bonanza episode, The Bloodline (December 31, 1960), along with 90 movie roles and 109 other television appearances over a 38-year span.
In the early 1980s he played John Peter McCallister, the "first Occidental to become a ninja" in NBC's The Master. The show was later featured on two episodes of the cult hit Mystery Science Theater 3000.
Lee Van Cleef died in Oxnard, California and was interred in Forest Lawn - Hollywood Hills Cemetery in Los Angeles. His gravestone says "Lee Van Cleef Jan 9, 1925 - Dec 16, 1989 'Best of the Bad' Love and Light".
Van Cleef was listed as one of the dedicatees at the end of Quentin Tarantino's 2004 film Kill Bill Vol. 2.