Abstract: Dynamin related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing reactive oxygen species (ROS) in cardiomyocytes. Drp1 is thus a promising therapeutic target for reducing cardiac ischemia/reperfusion (IR) injury. Lack of specificity of Drp1 small molecule inhibitors and the reliance on the use of Drp1 haploinsufficient hearts in genetic approaches have left the role of Drp1 in IR in question. We address these concerns using two approaches: a) Mice with short-term (3-week) conditional, cardiomyocyte specific Drp1 knock out (KO) and b) a novel, highy specific Drp1 GTPase inhibitor, Drpitor1a. Short-term Drp1 KO mice did not exhibit decreased exercise capacity or heart contractility, but isolated cardiac mitochondria demonstrated increased mitochondrial complex 1 activity, respiratory coupling, calcium retention capacity compared to controls. When exposed to IR injury on a Langendorff persusion system, Drp1 KO hearts had preserved contractility, decreased ROS, enchanced mitochondrial calcium capacity, increased resistance to mitochondrial permeability transition pore (MPTP) opening, and decreased cardiac ryanodine receptor (RYR2) gene expression. Phamacological inhibition of Drp1 with Drpitor1a following ischemia but prior to reperfusion was similarly protective of cardiac function and mitochondrial calcium homeostasis. In contrast to the benefits of short-term Drp1 inhibition, prolonged Drp1 ablation (6weeks) resulted in cardiomyopathy. We conclude that Drp1 is a key regulator of myocardial mitochondrial calcium handling and ROS generation following IR injury. Short-term inhibiton of Drp1 is a promising strategy for limiting early myocardial IR injury which is relevant for acute myocardial infarction, cardiac arrest, and heart transplantation.