Hydrogen sulfide (H2S) is a gasotransmitter with a variety of cardiovascular protective effects. 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- (ISO-) induced myocardial hypertrophy. We further investigated whether exogenous H2S supplement improved ISO-induced myocardial hypertrophy in a SIRT3-dependent manner. 10-week-old male 129S1/SvImJ (WT) mice and SIRT3 knockout (KO) mice were intraperitoneally injected with NaHS (50 μmol/kg/d) for two weeks and then intraperitoneally injected with ISO (60 mg/kg/d) for another two weeks. In WT mice, NaHS significantly reduced the cardiac index of ISO-induced mice, decreased the cross-sectional 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-AOC) 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-related protein 1 (DRP1) expression was downregulated. ERK, but not P38 and JNK, phosphorylation was downregulated. However, all above protective effects were unavailable in ISO-induced SIRT3 KO mice. Our present study suggested that exogenous H2S supplement inhibited ISO-induced cardiac hypertrophy depending on SIRT3, which might be associated with antioxidant stress.