Heart failure is a debilitating condition in which the heart cannot meet the metabolic demands of the body. Chronic β-adrenergic (β-AR) stimulation causes pathological myocardial remodeling that leads to heart failure, in part, by promoting apoptosis of cardiac myocytes. Work from our laboratory has shown that β-AR stimulated apoptosis is dependent on reactive oxygen species (ROS), but the molecular targets by which ROS mediate apoptosis is not known. One target of ROS that may contribute to activating the apoptosis pathway is the sarco-endoplasmic reticulum ATPase (SERCA2). SERCA2 is responsible for moving the large majority of intracellular calcium in the cardiac myocyte. We have identified that SERCA2 can undergo oxidative post-translational modification (OPTM) of cysteine C674: Low ROS increase activity while high ROS decreases. Since SERCA is the primary calcium transporter and is located in close proximity of the mitochondria, it is possible SERCA activity may affect the level of calcium in mitochondria, which in excess is a known activator of the intrinsic mitochondrial apoptosis pathway. Progressive loss of myocardial cells in ischemia and heart failure likely contributes to the pathogenesis of cardiomyopathy. We hypothesized that oxidation of SERCA2 at C674 increases mitochondrial calcium, thereby activating the mitochondrial apoptosis pathway. To address this thesis, we used a novel redox-insensitive SERCA2 mutation in which C674 is replaced by serine (C674S) to determine the role of oxidative inhibition of SERCA in H2O2-stimulated apoptosis in vitro. We tested our hypothesis using adult rat ventricular myocytes (ARVM) that overexpress wild type or SERCA C674 and assessed intra-organelle calcium content, mitochondrial function and activation of the apoptosis pathway. To measure mitochondrial calcium, we optimized the use of an ultrasensitive genetically-encoded calcium indicator (GECI) targeted to the mitochondria which was expressed in ARVM via adenovirus infection. Redox-insensitive SERCA C674S expressing ARVM displayed less sensitivity to H2O2-stimulated mitochondrial calcium uptake which was confirmed by measuring calcium sensitive pyruvate dehydrogenase phosphorylation status. Furthermore, SERCA C674S ARVM were protected from H2O2 -mediated apoptosis, indicated by a reduction in cytochrome c release and annexin V staining. Lastly, H2O2 treatment decreased the cytosolic ATP/ADP ratio and depolarized the mitochondrial membrane potential, however this was independent of SERCA C674 oxidation. Taken together, these experiments elucidate a novel role for SERCA2 activity in cardiac myocytes and provide a potential therapeutic target for reducing cardiac myocyte apoptosis, potentially improving cardiac function during heart failure.