Cave minerals deposited in the presence of microbes may host geochemical biosignatures that can be utilized to detect subsurface life on Earth, Mars, or other habitable worlds. The sulfur isotopic composition of gypsum (CaSO4·2H2O) formed in the presence of sulfur-oxidizing microbes in the Frasassi cave system, Italy, was evaluated as a biosignature. Sulfur isotopic compositions (δ34SV-CDT) of gypsum sampled from cave rooms with sulfidic air varied from -11 to -24‰, with minor deposits of elemental sulfur having δ34S values between -17 and -19‰. Over centimeter-length scales, the δ34S values of gypsum varied by up to 8.5‰. Complementary laboratory experiments showed negligible fractionation during the oxidation of elemental sulfur to sulfate by Acidithiobacillus thiooxidans isolated from the caves. Additionally, gypsum precipitated in the presence and absence of microbes at acidic pH characteristic of the sulfidic cave walls has δ34S values that are on average 1‰ higher than sulfate. We therefore interpret the 8.5‰ variation in cave gypsum δ34S (toward more negative values) to reflect the isotopic effect of microbial sulfide oxidation directly to sulfate or via elemental sulfur intermediate. This range is similar to that expected by abiotic sulfide oxidation with oxygen, thus complicating the use of sulfur isotopes as a biosignature at centimeter-length scales. However, at the cave room (meter-length) scale, reactive transport modeling suggests that the overall ∼13‰ variability in gypsum δ34S reflects isotopic distillation of circulating H2S gas due to microbial sulfide oxidation occurring along the cave wall-atmosphere interface. Systematic variations of gypsum δ34S along gas flow paths can thus be interpreted as biogenic given that slow, abiotic oxidation cannot produce the same spatial patterns over similar length scales. The expression and preservation potential of this biosignature is dependent on gas flow parameters and diagenetic processes that modify gypsum δ34S values over geological timescales. Key Words: Gypsum-Sulfur isotopes-Biosignature-Sulfide oxidation-Cave. Astrobiology 18, 59-72.