The dysfunction of endothelial progenitor cells (EPCs) was found to be associated with vascular complications in diabetes mellitus (DM) patients. Previous studies found that regular exercise could improve the function of EPCs in DM patients, but the underling mechanism was unclear. Irisin, a newly identified myokine, was induced by exercise and has been demonstrated to mediate some of the positive effects of exercise. In this study, we hypothesize that irisin may have direct effects on EPC function in DM mice. These data showed for the first time that irisin increased the number of EPCs in peripheral blood of DM mice and improved the function of EPCs derived from DM mice bone marrow. The mechanism for the effect of irisin is related to the PI3K/Akt/eNOS pathway. Furthermore, irisin was demonstrated to improve endothelial repair in DM mice that received EPC transplants after carotid artery injury. The results of this study indicate a novel effect of irisin in regulating the number and function of EPCs via the PI3K/Akt/eNOS pathway, suggesting a potential for the administration of exogenous irisin as a succedaneum to improve EPC function in diabetic patients who fail to achieve such improvements through regular exercise.