Cardiovascular complications are the major causes of death in patients with diabetes mellitus. Several studies have demonstrated that endothelial progenitor cells (EPCs), adult stem cells contributing to the regeneration of vascular endothelium, are dysfunctional under diabetic condition resulting in impaired peripheral circulation and delayed wound healing. In this study, we investigated the cellular alteration of EPCs under high glucose condition, to elucidate the mechanisms underlying diabetes-associated EPC dysfunction. EPCs were isolated from bone marrow and cultured in normal glucose (5.5 mM)- or high glucose (HG; 30 mM)-containing medium. High glucose treated-EPCs showed decreased ability to form tubule-like networks in Matrigel compared to EPCs under normal glucose, which matched well to the clinical observation of diabetic EPC dysfunction. Conversion of LC3-I to LC3-II was increased in EPCs under HG condition, showing that HG induced autophagy in EPCs. Flow cytometric analysis revealed generation of oxidative stress and disruption of mitochondrial permeability in HG exposed EPCs. Increased mitochondrial oxidative stress was also observed by mitochondria-specific superoxide indicator, MitoSOX(TM). Taken together, we demonstrated that autophagy and mitochondrial impairment were induced in EPCs under high glucose condition, giving a new insight into the mechanism underlying dysfunction of diabetic EPCs. We hope that our finding can contribute to the development of a new treatment option for cardiovascular complications in diabetic patients.