Aim To explore the mechanisms that heat shock proteins protected cardiomyocytes against apoptosis induced by hydrogen peroxide (H 2O 2). Methods The expression of heat shock protein 70 and αB crystallin in neonatal rat cardiomyocytes were induced by heat shock response (42℃, 1 h, and recovery for different durations). Cardiomyocyte apoptosis induced by 0.5 mmol/L hydrogen peroxide (H 2O 2) was determined by flow cytometric analysis. The activities of caspase 3, caspase 8, caspase 9 were assayed by caspase colorimetric assay kit and Western blot. The release of cytochrome C from mitochondria was observed by isolation of cellular components and Western blot. Results The expression of HSP70 and αB crystallin in neonatal rat cardiomyocytes significantly increased at 3 h and reached peak at 6～12 h after heat shock response. Heat shock response could inhibite H 2O 2 induced release of cytochrome c from mitochondria to cytoplasm, activation of caspase 3, caspase 8, caspase 9, and subsequent apoptosis in cultured neonatal rat cardiomyocytes. Conclusions HSPs protected cardiomyocytes from apoptosis by inhibiting the activation of both mitochondrial and death receptor pathways.