Aim To investigate the mechanisms of cardiomyocyte apoptosis during the oxidative stress. Methods Apoptosis of neonatal rat cardiomyocytes was induced by exposure to 0.5 mmol/L hydrogen peroxide (H 2O 2) for different durations and apoptosis was determined by terminal deoxynucleotidyl transferase mediated biotin dUTP nick end labeling (TUNEL). The activities of caspase 3, 8, 9 were assayed by caspase colorimetric assay kit and Western blot. The release of cytochrome C from mitochondria was observed by indirect immunofluorescence and Western blot. Results Exposure to 0.5 mmol/L hydrogen peroxide for 24 h resulted in neonatal rat cardiomyocyte apoptosis as shown by TUNEL positivity. The activities of caspase 3, 8, 9 were significantly increased after 4 h of hydrogen peroxide treatment, and reached peak at 8～12 h. The release of cytochrome C from mitochondria to cytoplasm was observed after 1～2 h treatment of hydrogen peroxide. Conclusions Hydrogen peroxide could induce the release of cytochrome C from mitochondria to cytoplasm, the activation of caspase 3, 8, 9 and subsequent apoptosis in neonatal rat cardiomyocytes, which means mitochondria and death receptor signal pathways are both involved in cardiomyocyte apoptosis induced by H 2O 2.