Our previous report has showed that the treatment of 48 h with 22 mM glucose prevents hypoxia-induced cardiac cell death. In the present study, we investigated whether high glucose affects the mitochondrial death pathway during hypoxia, and if it does, what relates to the high glucose induced cardioprotection. Heart-derived H9c2 cells were incubated in low (5.5 mM) or high (22 mM) glucose medium for 48 h, then transferred to a normoxic or hypoxic condition. The hypoxia-induced reduction of mitochondrial redox potential, assessed by MTT assay, was inhibited in high glucose treated cells. The mitochondrial membrane potential was significantly decreased by hypoxia in low glucose treated cells, but not in high glucose treated cells. The hypoxia-induced cytoplasmic accumulation of cytochrome c, released from the mitochondria, was blocked by a treatment of high glucose. High glucose did not induce the expression of an antiapoptotic protein Bcl-2, nor did it reduce a proapoptotic protein Bax, but it did inhibit a hypoxia-induced downregulation of Bcl-2. The cellular ATP contents were not changed by the treatment of high glucose for 48 h, and the hypoxia-induced decline of intracellular ATP level was observed in high glucose treated cells and in low glucose. A glycolytic inhibitor, 2-deoxyglucose, did not reverse the high glucose induced reduction of LDH release. The elevation of [ROS]_i induced by hypoxia was inhibited in high glucose treated cells. These results suggest that high glucose induced cardioprotection may be accounted for in part by the preservation of MMP and the maintenance of a basal level of [ROS]_i during hypoxia.