Calreticulin (CRT), a Ca²⁺-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H2O2, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca²⁺ ([Ca²⁺]i) were significantly increased by H2O2, whereas in controls, only a slight increase was observed. The H2O2-induced apoptosis was enhanced by the increase in [Ca²⁺]i caused by thapsigargin in control cells, but was suppressed by 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester, a cell permeable Ca²⁺ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca²⁺]i in the sensitivity to the H2O2-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared to controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca²⁺]i in the CRT-overexpressing cells treated with H2O2, compared with controls. Thus, we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H2O2 through a change in Ca²⁺ homeostasis and the regulation of the Ca²⁺/calpain/caspase-12 pathway in myocardiac cells.