Calnexin (CNX) is an endoplasmic reticulum (ER) quality control chaperone that has been implicated in ER stress. ER stress is a prominent pathological feature of various pathologic conditions, including cardiovascular diseases. However, the role of CNX and ER stress has not been studied in the heart. In the present study, we aimed to characterize the role of CNX in cardiomyocyte physiology with respect to ER stress, apoptosis, and cardiomyocyte Ca(2+) cycling. We demonstrated significantly decreased CNX mRNA and protein levels by LentiVector mediated transduction of targeting shRNAs. CNX silenced cardiomyocytes exhibited ER stress as evidenced by increased GRP78 and ATF6 protein levels, increased levels of spliced XBP1 mRNA, ASK-1, ERO1a, and CHOP mRNA levels. CNX silencing also led to significant activation of caspases-3 and -9. This activation of caspases was associated with hallmark morphological features of apoptosis including loss of sarcomeric organization and nuclear integrity. Ca(2+) imaging in live cells showed that CNX silencing resulted in Ca(2+) transients with significantly larger amplitudes but decreased frequency and Ca(2+) uptake rates in the basal state. Interestingly, 5 mM caffeine stimulated Ca(2+) transients were similar between control and CNX silenced cardiomyocytes. Finally, we demonstrated that CNX silencing induced the expression of the L-type voltage dependent calcium channel (CAV1.2) but reduced the expression of the sarcoplasmic reticulum ATPase (SERCA2a). In conclusion, this is the first study to demonstrate CNX has a specific role in cardiomyocyte viability and Ca(2+) cycling through its effects on ER stress, apoptosis and Ca(2+) channel expression.