Polysulfides commonly occur in anaerobic, microbial active environments, where they play key roles in sulfur cycling and redox transformations. Anaerobic survival of microorganisms requires the formation of protein disulfide bond (DSB). The relationship between polysulfides and anaerobic DSB formation has not been studied so far. Herein, we discovered that polysulfides can efficiently mediate protein DSB formation of microorganisms under anaerobic conditions. We used polysulfides to treat proteins, including roGFP2, Trx1, and DsbA, under anaerobic conditions and found that all three proteins formed intramolecular DSB