Aldose reductase (AR), a member of the aldo-keto reductase family, has been demonstrated to play a central role in mediating myocardial ischemia-reperfusion (I/R) injury. Recently, using transgenic mice broadly over expressing human aldose reductase (ARTg), we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects heart from I/R injury. To rigorously delineate mechanisms by which AR pathway influences myocardial ischemic injury, we investigated the role played by ROS, antioxidant enzymes and mitochondrial permeability transition MPT pore opening in hearts from ARTg or littermates (WT) subjected to I/R. MPT pore opening after I/R was determined using mitochondrial uptake of 2-deoxyglucose (2-DG) ratio, while H2O2 was measured as a key indicator of ROS. Myocardial 2-DG uptake ratio and calcium induced swelling was significantly greater in mitochondria from ARTg mice than in WT mice. Blockade of MPT pore with cyclosphorin A during I/R reduced ischemic injury significantly in ARTg mice hearts. H2O2 measurements indicated mitochondrial ROS generation after I/R was significantly greater in ARTg mitochondria than in WT mice hearts. Furthermore, the levels of antioxidant GSH were significantly reduced in ARTg mitochondria than in WT. Resveratrol treatment or pharmacological blockade of AR significantly reduced ROS generation and MPT pore opening in mitochondria of ARTg mice hearts exposed to I/R stress. This study demonstrates that MPT pore opening is a key event by which aldose reductase pathway mediates myocardial ischemia-reperfusion injury and that the MPT pore opening after I/R is triggered, in part, by increases in reactive oxygen species generation in ARTg mice hearts. Therefore, inhibition of aldose reductase pathway protects mitochondria and hence, may be a useful adjunct for salvaging ischemic myocardium.