An increase in cytosolic Ca²⁺ via capacitative calcium entry mediated pathway, attributed to members of the transient receptor potential (TRP) superfamily, TRPC1 and TRPC3, have been reported to play an important role in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca²⁺ also plays a critical role in mediating cell death in response to ischemia/reperfusion (I/R). Therefore, we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Cardiomyocytes isolated from wild-type (WT) mice and from mice over expressing TRPC3 in the heart were subject to 90 min ischemia and 3hr reperfusion. After I/R, viability was 51±1% in WT and 42±5% in TG (p<0.05). Apoptosis assessed by Annexin-V was significantly increased in TRPC3 group compared to WT (32±1% vs 21±3%; p<0.05); however, there was no difference in necrosis between groups. Treatment of TRPC3 cells with the CCE inhibitor SKF96365 (0.5µM), significantly improved cellular viability (54±4%) and decreased apoptosis (15±4%); in contrast, the L-type Ca²⁺ channel inhibitor verapamil (10µM) had no effect. Calpain-mediated cleavage of fodrin was 3-fold higher in the TG group following I/R compared to WT (p<0.05); this was significantly attenuated by SKF96365. The calpain inhibitor, PD150606 (25µM) attenuated the increase in both -fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca²⁺ overload stress, but did not affect the response to TNF- induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, by increased calpain activation.