Endothelial cells (ECs) maintain the structure and function of blood vessels and are readily exposed to exogenous and endogenous toxic substances in the circulatory system. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the blood and differentiate to EC, which are known to participate in angiogenesis and regeneration of injured vessels. Dysfunction in EPC contributes to cardiovascular complications in patients with diabetes, but the precise molecular mechanisms underlying diabetic EPC abnormalities are not completely understood. The aim of this study was to investigate the mechanisms underlying diabetic EPC dysfunction using methylglyoxal (MG), an endogenous toxic diabetic metabolite. Data demonstrated that MG decreased cell viability and protein expression of vascular endothelial growth factor receptor (VEGFR)-2 associated with functional impairment of tube formation in EPC. The generation of advanced glycation end (AGE) products was increased in EPC following exposure to MG. Blockage of receptor for AGE (RAGE) by FPS-ZM1, a specific antagonist for RAGE, significantly reversed the decrease of VEGFR-2 protein expression and angiogenic dysfunction in MG-incubated EPC. Taken together, data demonstrated that MG induced angiogenic impairment in EPC via alterations in the AGE/RAGE-VEGFR-2 pathway which may be utilized in the development of potential therapeutic and preventive targets for diabetic vascular complications.