Aim To observe the effects of captopril late preconditioning on intracellular calcium concentration ([Ca 2+ ]i) in cardiac myocytes of the neonatal rat undergone anoxia-reoxygenation injury and evaluate its delayed protective role. Methods The anoxia-reoxygenation models in cultured ventricular myocytes of neonatal rat were established and divided into 7 groups: normal, anoxia-reoxygenation,preconditioning, captopril,captopril + PKC inhibitor,captopril + NOS inhibitor(L-NAME), captopril + NF-κB inhibitor(PDTC), Using spectrophotometry, the activities of malondialde-hyde (MDA)and superoxide disputase (SOD) were observed. The lactate dehydrogenase (LDH) were observed by biochemical instruments. Using Flou-3 /AM loading and flow cytometry technique, the [Ca 2+ ]i were observed. Results [Ca 2+ ]i was increased in anoxia-reoxygenation group (789±9 nmol/L vs. 414±37 nmol/L, p<0.01). The precondition of captopril and anoxia- reoxygenation resulted in a significant decrease in [Ca 2+ ]i(594±5 nmol/L, 507±32 nmol/L vs. 789±9 nmol/L, p<0.01), at one time [Ca 2+ ]i were lightly increased than normal oxygen condition(p<0.05). Before treatment with captopril, we add PKC blocking agent, PDTC, L-NAME to cell media respectively, captopril late preconditioning relieved calcium overload during anoxia-reoxygenation condition, myocytes [Ca 2+ ]i were measured as 676±32 nmol/L,700±37 nmol/L, 689±11 nmol/L, which were significantly increased compared with captopril group(p<0.05); while compared with anoxia-reoxygenation group were still reduced(p<0.05). Conclusion Captopril late preconditioning in cardiac myocytes is triggered by slightly increasing [Ca 2+ ]i and weakening the calcium overload and lipid peroxidation effect during subsequenced anoxia-reoxygenation injury. It may include the route of PKC, NF-κB, NOS.