Aims: Clinical use of cisplatin (Cisp), one of the most widely used, common, and effective chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Previous studies suggest that hydrogen sulfide (H2S) alleviates Cisp-induced acute kidney injury (AKI). However, the underlying mechanism remains largely unclear. Results: A single intraperitoneal injection of Cisp is employed to induce AKI, and the mice exhibit severe kidney dysfunction and histological damage at day 4 after Cisp injection. Here, we reported that H2S alleviated Cisp-caused renal toxicity via SIRT3 activation and subsequent improvement of mitochondrial ATP production. Using a biotin-switch assay, we showed that H2S increased S-sulfhydration of SIRT3 and induced deacetylation of its target proteins (OPA1, ATP synthase β, and superoxide dismutase 2). These effects of H2S were associated with a reduction of mitochondrial fragmentation, an increase in ATP generation, and less oxidative injury. Notably, the S-sulfhydration of SIRT3 induced by H2S was abrogated when Cys256, Cys259, Cys280, and Cys283 residues on SIRT3 (two zinc finger domains) were mutated. Innovation and Conclusion: Our data suggest that H2S attenuates Cisp-induced AKI by preventing mitochondrial dysfunction via SIRT3 sulfhydrylation VSports手机版. Antioxid. Redox Signal. 31, 1302-1319. .