Mitochondrial fission protein, dynamin-related protein-1 (DRP-1), may be a key determinant of cardiac resistance to ischemia-reperfusion (I-R) and other stresses. We examined the impact of I-R on myocardial expression of DRP-1 (together with Akt and Erk1/2), mitochondrial respiration, and injury in I-R hearts in 4mo male C57Bl/6 mice subject to DRP-1 inhibition. Functional recoveries of Langendorff perfused hearts subjected to 25 min ischemia/45 min reperfusion were significantly improved by pre-treatment with 1 µM MDIVI-1. Cell death (LDH efflux) was only modified by a higher 5 µM concentration. Protective effects were not replicated by an alternate inhibitor dynasore hydrate (1 µM). Ischemic insult repressed mitochondrial O2 consumption during early reperfusion, with a ~50% fall in complex I activity that was effectively reversed by MDIVI-1. Functional benefits were associated with MDIVI-1 dependent reductions in mitochondrial DRP-1 expression and cytosolic Erk1/2 phosphorylation during I-R, while phosphorylation of cardiac Akt was significantly augmented by MDIVI-1. A similar pattern was evident in parallel studies of H2O2 (400 µM) challenged H9c2 myoblasts, with MDIVI-1 negating oxidative-stress Erk1/2 phosphorylation whereas phosphoactivation of Akt was preserved. These data support a key role for DRP-1 in repressing I-R tolerance, mitochondrial function, and protective Akt phosphorylation (whilst also augmenting Erk1/2 activation). This role of DRP-1 in control of stress-resistance, mitochondrial function and survival-kinase signalling supports the potential value of targeting the protein to protect ischemic myocardium