Objective:To investigate the direct effect of different concentrations of hydrogen sulfide(H_2S)on neonatal rat myocardial cells by hypoxia injury during different time point,and the indirect effect of reoxygenation injury in order to analyze the protective role of H_2S on myocardial cells subjected to hypoxia/reoxygenation injury. Methods:Primary cultured myocardial cells from neonatal rats were randomly divided into 9 groups:Normal control group,Hypoxia NaHS 0 μmol/L group,100 μmol/L group,200 μmol/L group,400 μmol/L group and those groups subjected to hypoxia for 24 h,48 h and 72 h. Hypoxia/reoxygenation NaHS 0 μmol/L group,100 μmol/L group,200 μmol/L group,400 μmol/L group and those groups subjected to hypoxia for 24 h,48 h,72 h and then reoxygenation for 2 h. The number of survival cells were counted and the activity of lactate dehydrogenase(LDH)were measured respectively at each time point. Results:At the same time point of 24 h,48 h,72 h hypoxia,the number of survival cells increased and the activity of LDH decreased in hypoxia NaHS treatment groups as compared with Hypoxia NaHS 0 μmol/L group(P<0.01). The protective role of Hypoxia NaHS 200 μand 400 μmol/L groups were better than NaHS 100 μmol/L group at 24 h of time poit(P<0.05～0.01). The number of survival cells by hypoxia/reoxygenation increased and the leakage of LDH from reoxygenation cells decreased in hypoxia/reoxygenation NaHS treatment groups as compared with hypoxia/reoxygenation NaHS 0 μmol/L group(P<0.01).Conclusion:NaHS 100 to 400 μmol/L had the protective effects on myocardial cells by hypoxia/reoxygenation injury. NaHS 200 to 400 μmol/L might offer the best protective effects on myocardial cells by hypoxia injury at 24 h.