Abstract: To study the effects of Roudoukou-8 San against hydrogen peroxide-induced cardiomyocyte in neonatal rats and to explore its mechanism. Cardiomyocytes were isolated and cultivated by neonatal rats and the injure models were established by H₂O₂. Methyl thiazolyl tetrazolium(MTT)assay was used to detect the protective effects of Roudoukou-8 San on H₂O₂-induced cardiomyocyte. The effects of Roudoukou-8 San on myocardium morphology were observed under inverted microscope. The contents of lactate dehydrogenase(LDH), creatine kinase(CK)and aspartate aminotransferase(AST)in cell culture medium were determined by Automatic biochemical instrument; The levels of malonydialdehyde(MDA), superoxide dismutase(SOD)and NO in the cells were detected by kit method. The apoptotic morphology of cardiomyocytes was observed by Hoechst fluorescence staining. Cell apoptosis were measured by Annexin V and PI double staining and flow cytomety. 100μmol/L H₂O₂ for 2 h could cause about 50% of myocardial injury. In the inverted optical microscope, H₂O₂ model group showed increased cell gap, decreased cell count, cell cytoplasmic vacuoles and other obvious damage. Roudoukou-8 San protected cell from H₂O₂-induced morphlogical improved in different degrees, reduced the release of LDH, CK and AST content, reduced the content of MDA, NO in myocardial cells significantly and increased the activity of SOD. Roudoukou-8 San energy significantly inhibited H₂O₂ damage myocardial cell apoptosis. Our study suggested that Roudoukou-8 San can protect cardiomyocyte from H₂O₂-induced injury by improving the cell viability, reducing oxidative stress injury, inhibiting inflammatory reaction and inhibiting cell apoptosis.