Ischemia-reperfusion (IR) induced calcium overload and production of reactive oxygen species (ROS) can trigger apoptosis by promoting the release of pro-apoptotic factors via the mitochondrial permeability transition pore (mtPTP). While it is clear that endurance exercise provides cardioprotection against IR induced injury, it is unknown if exercise training directly alters mitochondria phenotype and confers protection against apoptotic stimuli in both subsarcolemmal (SS) or intermyofibrillar (IMF) mitochondria. We hypothesized that exercise training increases expression of endogenous antioxidant enzymes and other anti-apoptotic proteins resulting in a SS and IMF mitochondrial phenotype that resists apoptotic stimuli. Mitochondria isolated from hearts of sedentary (n = 8) and exercised trained (n = 8) adult, male rats were studied. Endurance exercise increased the protein levels of primary antioxidant enzymes in both SS and IMF mitochondria. Further, exercise increased the levels of anti-apoptotic proteins in the heart including the apoptosis repressor with a caspase recruitment domain and inducible heat shock protein 70. Importantly, our findings reveal that endurance exercise training attenuates ROS-induced cytochrome c release from heart mitochondria. These changes are accompanied by a lower maximal rate of mtPTP opening (Vmax) and prolonged time to Vmax in both SS and IMF cardiac mitochondria. These novel findings reveal that endurance exercise promotes biochemical alterations in cardiac SS and IMF mitochondria resulting in a phenotype that resists apoptotic stimuli. Further, these results are consistent with the concept that exercise-induced mitochondrial adaptations contribute to exercise-induced cardioprotection.