Objective To investigate the preventive effects of total saponin from Tribulus terrestris (GSTT) on cardiomyocytes subjected to low oxygen tension and re-oxygenation injury in vitro. Methods First passage of cultured cardiomyocytes was subjected to hypoxia followed by oxygenatim. The cells were divided into 4 groups, namely normal group, injury group, and large and low dose of GSTT groups. The frequency of contraction, survival rate and release of myocardial enzymes were determined. The morphologic changes were observed with light and electron microscopy. Results Both large and low dose of GSTT significantly improved the survival rate of cardiomyocytes after hypoxia and re-oxygenation injury (P<0.01, P<0.05), and the drug inhibited the release of myocardial enzymes (P<0.01, P<0.05 ). The effect of large dose was superior to that of low dose. In addition, GSTT could improve the contractile behavior of cardiomyocytes after injury (P<0.01), and it could also ameliorate the ultrastructural injuries. Conclusion GSTT could protect cultured neonatal rat cardiomyocytes from hypoxia/ reoxygenation injury.