Vaspin is an adipocytokine recently identified in the visceral adipose tissue of diabetic rats and having anti-diabetic effects. We have recently shown that vaspin has anti-atherogenic effect through Akt-mediated inhibition of endothelial cell apoptosis. Decreased activity of endothelial nitric oxide synthase (eNOS) plays an important role in the pathogenesis of atherosclerosis. Asymmetric dimethylarginine (ADMA) is a well-known endogenous competitive inhibitor of eNOS and risk factor of cardiovascular diseases. The aim of this study was to examine whether vaspin might protect against atherosclerosis through its beneficial effects on the ADMA-eNOS system. Treatment of vaspin significantly increased NO secretion from endothelial cells and isolated aorta from Sprague-Dawley (SD) rats. Furthermore, treatment of vaspin prevented fatty acid-induced decrease in endothelium-dependent vasorelaxation in isolated aorta of SD rat. For the mechanism of vaspin-induced NO biosynthesis, vaspin activated the STAT3 signaling pathway and stimulated eNOS phosphorylation (Ser 1177), a marker of eNOS activation, through STAT3-dependent mechanism. Furthermore, vaspin treatment increased the expression of dimethylarginine dimethylaminohydrolase (DDAH) II, the responsible enzyme for the degradation of ADMA, leading to a reduction in ADMA levels. Vaspin-induced increase in DDAH II gene expression was through STAT3-mediated stimulation of DDAH II promoter activity. These results suggest that vaspin increases eNOS activity by reducing ADMA level through STAT3-mediated regulation of DDAH II expression. Our findings provide a novel molecular mechanism of antiatherogenic actions of vaspin.