Polysulfides is reactive persulfide species which have extra sulfur atoms in cysteine thiol and cellular proteins contain polysulfides abundantly (protein polysulfuration). In this study, we investigated the translation-coupled mechanism of protein polysulfidation mediated by cysteinyl-tRNA synthetase (CARS) and its biological functions. CARSs from various biological species including bacteria and mammals are found to have cysteine persulfide-producing activity and to be critically involved in protein polysulfuration. Protein polysulfuration is suggested to play important roles in the regulation of cellular function such as mitochondrial morphogenesis.