Methylmercury (MeHg) reacts with nucleophilic sulfur species to form sulfur adducts, such as the low-toxic metabolite bismethylmercury sulfide [(MeHg)₂S]. We found that protein-bound persulfides interact with MeHg to form (MeHg)₂S and identified glutathione S-transferase pi 1 as a S-sulfhydrated protein involved in (MeHg)₂S formation. Although glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a house-keeping protein abundantly expressed in various tissues, has been reported to undergo S-sulfhydration in the presence of sulfur donors or cystathionine γ-lyase, the biological significance of this post-translational modification is poorly understood. In this study, we investigated the possible interaction between GAPDH and MeHg to form (MeHg)₂S. High-performance liquid chromatography/atomic absorption spectrophotometry revealed that (MeHg)₂S was formed during the reaction of MeHg with a model of cysteine persulfide and GAPDH following incubation of the protein with NaHS. After reacting with NaHS, GAPDH C152S and C156S mutants transformed MeHg into (MeHg)₂S, whereas formation of the sulfur adduct was not observed for the C247S mutant, suggesting that Cys247 is critical for conversion of MeHg to (MeHg)₂S. These results suggest that the sulfane sulfur on Cys247 of GAPDH plays a protective role in reducing MeHg toxicity.