This study was aimed at investigating the protective effect and mechanism of vitexin preconditioning (VPC) on cultured neonatal rat cardiomyocytes after anoxia and reoxygenation (A/R). An A/R model was established by using cultured neonatal rat cardiomyocytes. Cellular injury was evaluated by measuring cell viability, the releases of creatine kinase (CK), and lactate dehydrogenase (LDH). The apoptosis rate of cardiomyocytes after Anoxia/reoxygenation and the activities of extracellular signal-regulated protein kinases (ERKs) were measured. The intracellular calcium indicated by the fluorescence in cardiomyocytes was measured by the laser confocal microscope. Vitexin preconditioning (10, 30 and 100 μM) significantly enhanced the cell viability, markedly inhibited A/R-induced increases of LDH and CK release, obviously decreased the number of apoptotic cardiomyocytes and markedly decreased the fluorescence intensity value of [ Ca2+]iin cardiomyocytes. Exposure to anoxia or vitexin preconditioning significantly increased the phospho-ERK level, and the increase was markedly inhibited by PD98059, an inhibitor of the upstream kinase of ERK. These results suggest that vitexin preconditioning has a protective effect on cardiomyocytes A/R injury through the improvement of cell viability, decrease of LDH and CK release, such that the protective mechanism may relate to its ability to inhibit the cardiomyocytes apoptosis, reduce the cardiomyocytes calcium overload and increase the abundance of phosphor-ERK1/2 of the cardiomyocytes after anoxia and reoxygenation.