Mammoth Cave (Kentucky, USA) is the world’s longest human-navigable cave system. Gypsum (CaSO4•2H2O) crystals line many dry passages of the limestone cave and the source of sulfur for the gypsum remains uncertain. Previous workers have suggested sulfate from pyrite oxidation, Mississippian-aged sulfate evaporites, and Pleistocene soil sulfate as possible sulfur sources. We use sulfur isotopes (δ34S) to constrain the gypsum sulfur source by comparing the δ34S values of different possible sources to that of gypsum throughout the cave. δ34Sgypsum values (n = 106) from 12 different locations within the Mammoth Cave system are along a continuum of δ34S values from −12.0‰ to +12.8‰, with little variability along the crystal growth axis or between samples within the same cave chamber. Neither sulfur from coeval sedimentary evaporites (i.e., gypsum, anhydrite) nor from formations overlying the cave is required to explain the δ34Sgypsum data. Rather, the range of pyrite δ34S in strata immediately surrounding the cave is sufficient to generate the spectrum of observed δ34Sgypsum. Modern water δ34SSO4 values are similar to the host formation δ34Spyrite, suggesting that oxidized pyrite from the host formation continues to be a major sulfur source to this day. Together, these observations strongly suggest a significant local source of sulfur for Mammoth Cave gypsum, the majority (66%–100%) of which is derived from the oxidation of pyrite in strata adjacent to the cave.