Dysfunction of mitochondrial calcium homeostasis transforms this cation from a key regulator of mitochondrial function, into a death effector during post-ischemic reperfusion. High intramitochondrial calcium and prevailing cellular conditions favor the opening of the mitochondrial permeability transition pore (mPTP), that induces mitochondrial swelling and provides a mechanism for cytochrome c release, a hallmark signal protein of the mitochondrial apoptosis pathway; indeed, a second mechanism induced by pro-apoptotic BAX protein, could account for cytochrome c leak in the post-ischemic heart. The present study was undertaken to determine which one of these mechanisms triggers the mitochondrial apoptosis pathway in the reperfused heart. To accomplish this goal we prevented the opening of the mPTP in such hearts, by diminishing calcium overload with Ru360, a specific mitochondrial calcium uniporter inhibitor. We found that mPTP opening in reperfused hearts increased along with reperfusion time and concurs with cytochrome c release from mitochondria. Maximal cytochrome c release correlated with mitochondrial dysfunction and complete NAD+ deletion. Fully inserted BAX was detected early after reperfusion and remained unchanged during the evaluated reperfusion times. Remarkably, heart perfusion with Ru360, inhibited mPTP opening and BAX docking into the mitochondrial membranes, suggesting a mPTP upstream role on BAX migration/insertion.