Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein Drp1 inhibition, attenuates I/R injury through modifications in mitochondrial metabolism. Rats were subjected to ischemia-reperfusion (I/R) through coronary artery ligation and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area and mitochondrial morphology where determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, while cardiac function after I/R was improved in Drp1K38A-treated rats compared to controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and upon I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.