Improving the dysfunction of endothelial progenitor cell (EPCs) in patients with diabetes mellitus is important for preventing vascular complication. Vaspin, an adipocytokine, has the anti-atherogenic properties rely on its positive effect on nitric oxide (NO) bioavailability. We hypothesis that vaspin may ameliorate high glucose induced dysfunction of EPCs. In rat bone marrow derived EPCs, glucose treatment results in a decrease in the proliferation and migration capacity in a dose dependent manner. These detrimental effects can be alleviated by vaspin. Furthermore, vaspin increased the production of NO and the effect of vaspin on EPCs can be diminished partly by the eNOS inhibitor (L-NAME). We assessed total eNOS protein expression and Ser(1177)-phospho-eNOS expression and found that vaspin not only induced eNOS protein expression but also up regulate the eNOS activation. Subsequently, we investigated protein kinase B (Akt) activation in the presence and absence of phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor (LY-2940002). Vaspin increased total Akt and Ser(473)-phospho-Akt expression and these effects can be blocked by LY-2940002. The results of our study indicate a novel effect of vaspin to regulate eNOS expression and function in EPCs via a PI3K/Akt/eNOS pathway; vaspin may have a protective effect in patients with diabetes to prevent the occurrence of vascular complication.