Cardiac stem cell (CSC) therapy is a promising approach to treat ischemic heart disease. However, the poor survival of transplanted stem cells in the ischemic myocardium has been a major impediment in achieving an effective cell-based therapy against myocardial infarction. Inhibiting mitochondrial fission has been shown to promote survival of several cell types. However, the role of mitochondrial morphology in survival of human CSC remains unknown. In this study, we investigated whether mitochondrial division inhibitor-1 (Mdivi-1), an inhibitor of mitochondrial fission protein dynamin-related protein-1 (Drp1), can improve survival of a novel population of human W8B2+ CSCs in hydrogen peroxide (H2O2)-induced oxidative stress and simulated ischemia-reperfusion injury models. Mdivi-1 significantly reduced H2O2-induced cell death in a dose-dependent manner. This cytoprotective effect was accompanied by an increased proportion of cells with tubular mitochondria, but independent of mitochondrial membrane potential recovery and reduction of mitochondrial superoxide production. In simulated ischemia-reperfusion injury model, Mdivi-1 given as a pretreatment or throughout ischemia-reperfusion injury significantly reduced cell death. However, the cytoprotective effect of Mdivi-1 was not observed when given at reperfusion. Moreover, the cytoprotective effect of Mdivi-1 in the simulated ischemia-reperfusion injury model was not accompanied by changes in mitochondrial morphology, mitochondrial membrane potential, or mitochondrial reactive oxygen species production. Mdivi-1 also did not affect mitochondrial bioenergetics of intact W8B2+ CSCs. Taken together, these experiments demonstrated that Mdivi-1 treatment of human W8B2+ CSCs enhances their survival and can be employed to improve therapeutic efficacy of CSCs for ischemic heart disease.