Oxidative stress is a crucial factor inducing cardiomyocyte apoptosis due to cardiac hypertrophy. Additional evidence has revealed that H2S plays an antioxidant role and is cytoprotective. Hence, we aimed to elucidate whether H2S prevents cardiomyocyte apoptosis due to cardiac hypertrophy via its antioxidant function. The cardiac hypertrophy model was obtained by injecting a high dose of isoproterenol (ISO) subcutaneously, and the hemodynamic parameters were measured in groups that received either ISO or ISO with the treatment of NaHS. TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling) and EM (electron microscopy) experiments were performed to determine the occurrence of apoptosis in heart tissues. The expression of caspase-3 protein in the cytoplasm and NADPH oxidase 4 (NOX4), and cytochrome c (cyt c) proteins in the mitochondria were analyzed using Western blotting. In contrast, to determine whether ISO-induced apoptosis in the cultured cardiomyocytes may be related to oxidative stress, JC-1 and MitoSOX assays were performed to detect the mitochondrial membrane potential and reactive oxygen species (ROS) production in the mitochondria. Exogenous H2S was found to ameliorate cardiac function. The histological observations obtained from TUNEL and EM demonstrated that treatment with NaHS inhibited the occurrence of cardiac apoptosis and improved cardiac structure. Moreover, H2S reduced the expression of the cleaved caspase-3, NOX4 and the leakage of cyt c from the mitochondria to the cytoplasm. We also observed that exogenous H2S could maintain the mitochondrial membrane potential and reduce ROS production in the mitochondria. Therefore, H2S reduces oxidative stress due to cardiac hypertrophy through the cardiac mitochondrial pathway.