Objective: Effects of intra-erythrocyte calcium content on the appearance and function of erythrocyte during cardiopulmonary bypass (CPB) remain unknown. We therefore aimed to compare the change of intra-erythrocyte Ca2+ content in pediatric patients before and after CPB. Methods: We enrolled 26 pediatric patients undergoing surgical repair of ventricular or atrial sepal defect under CPB in this prospective study. Heparinized venous blood samples were taken from the central vein before, at the beginning and after CPB. The concentrations of intra-erythrocyte Ca2+ were measured by fluorescent probes (Fluo-3-AM), along with plasma Ca2+. Results: There were no significant differences in the levels of post-CPB intra-erythrocyte Ca2+ content compared with those before CPB. At the beginning of CPB, plasma Ca2+ decreased significantly compared with that before CPB. Conclusion: The findings indicate that CPB doesn’t change the appearance and function of erythrocyte.