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Sirtuin 3 (SIRT3) is closely related to mitochondrial function and oxidative stress. We found that NaHS increased SIRT3 expression in the preventive effect on isoproterenol\u2010 (ISO\u2010) induced myocardial hypertrophy. We further investigated whether exogenous H<jats:sub>2<\/jats:sub>S supplement improved ISO\u2010induced myocardial hypertrophy in a SIRT3\u2010dependent manner. 10\u2010week\u2010old male 129S1\/SvImJ (WT) mice and SIRT3 knockout (KO) mice were intraperitoneally injected with NaHS (50\u2009<jats:italic>\u03bc<\/jats:italic>mol\/kg\/d) for two weeks and then intraperitoneally injected with ISO (60\u2009mg\/kg\/d) for another two weeks. In WT mice, NaHS significantly reduced the cardiac index of ISO\u2010induced mice, decreased the cross\u2010sectional area of cardiomyocytes, and inhibited the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA. The activity of total antioxidant capacity (T\u2010AOC) and superoxide dismutase (SOD) in the myocardium was increased, but the level of malondialdehyde (MDA) was decreased. The fluorescence intensity of dihydroethidium staining for superoxide anion was attenuated. Optic atrophy 1 (OPA1) expression was upregulated, while dynamin\u2010related protein 1 (DRP1) expression was downregulated. ERK, but not P38 and JNK, phosphorylation was downregulated. However, all above protective effects were unavailable in ISO\u2010induced SIRT3 KO mice. 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