Aim To study the cardioprotective mechanism of hydrogen sulfide(H2S) on oxidative stress induced by hydrogen peroxide. Methods Primary-cultured cardiomyocytes were obtained from neonatal rats,an oxidative stress injury model was established by exposing the cells to H2O2 for 2 h.To study the cardioprotective mechanism of hydrogen sulfide(H2S) on oxidative stress induced by hydrogen peroxide,the cells were pre-incubated with ly294002 for 30 min.The morphology change of cardiomyocytes was observed by inverted phase contrast microscope,the activities of lactate dehydrogenase(LDH) and superoxide dismutase(SOD),and content malondialdehyde(MDA) were determined,the activation of the PI3K/Akt signal pathway was observed by Western blotting,the cardiomyocytes apoptosis was detected by AnnexinV/PI flow cytometry. Results After exposing to H2O2 for 2 h,the release of LDH,MDA and apoptosis were increased,the activities of SOD and ratio of Bcl-2/Bad were decreased(P<0.05).After pretreated with sodium hydrosulfide(NaHS) for 30 mins,the injury effect was attenuated,moreover,the phosphorylation of Akt,Bad and ratio of Bcl-2/Bad were upregulated.LY294002 could block the protection effect of NaHS partly. Conclusion H2S can attenuate hydrogen peroxide-induced rat neonatal cardiomyocytes injury,which may involve the PI3K/Akt pathway.