The gun, which the auction house Bonhams says Hickok was carrying when he died, is expected to fetch $300,000 to $500,000.
Hickok was playing poker in Deadwood, S.D., on Aug. 2, 1876, and holding the infamous "dead man's hand" of aces and eights when he was shot to death by Jack McCall.
Hickok was known as the best shot in the West. The gun up for auction has a .32 rimfire, six-inch barrel and finished rosewood grips. It comes with documentation proving its provenance, Bonhams said, including details of how it was passed through multiple generations in Deadwood to the current owner's father.
The auction is scheduled for Nov. 18.