Exogenous ubiquitin (UB) plays a protective role in β-AR-stimulated and ischemia/reperfusion (I/R)-induced myocardial remodeling. Here, we report that UB treatment inhibits hypoxia/reoxygenation (H/R)-induced apoptosis in adult rat ventricular myocytes (ARVMs). Activation of Akt was higher, while activation of GSK-3β was lower in UB-treated cells post-H/R. Oxidative stress was lower, while the number of ARVMs with polarized mitochondria was significantly greater in UB-treated samples. ARVMs express CXCR4 with majority of CXCR4 localized in the membrane fraction. CXCR4 antagonism using AMD3100, and siRNA-mediated knockdown of CXCR4 negated the protective effects of UB. Two mutated UB proteins (unable to bind CXCR4) had no effect on H/R-induced apoptosis, activation of Akt and GSK-3β or oxidative stress. UB treatment enhanced mitochondrial biogenesis, and inhibition of mitochondrial fission using mDivi1 inhibited H/R-induced apoptosis. Ex vivo, UB treatment significantly decreased infarct size and improved functional recovery of the heart following global I/R. Activation of caspase-9, key player of mitochondrial death pathway, was significantly lower in UB-treated hearts post-I/R. UB, most likely acting via CXCR4, plays a protective role in H/R-induced myocyte apoptosis and myocardial I/R injury via the modulation of mitochondrial homeostasis and mitochondrial death pathway of apoptosis.