Aim Visceral adipose tissue‐derived serine protease inhibitor (vaspin) is an adipocytokine with insulin‐sensitizing activity originally identified in visceral adipose tissues of obesity‐related type II diabetic rats. We previously showed that vaspin inhibits vascular cell migration and apoptosis as well as inflammatory responses, which are crucial for the development of hypertension. However, little is known about the effects of vaspin on vascular reactivity. The aim of this study was thus to explore the effects of vaspin on contraction and relaxation of isolated blood vessel. Methods After mesenteric arteries were isolated from male Wistar rats, the effects of pretreatment with vaspin (3 ng mL−1, 30 min) on concentration–contraction and concentration–relaxation relationships for each agent were examined. The effects of vaspin on acetylcholine (ACh)‐induced phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and ACh esterase (AChE) activity were also examined using Western blotting and colorimetric method respectively. Results Vaspin did not affect noradrenaline‐ or 5‐hydroxytryptamine‐induced contraction. In contrast, vaspin augmented ACh‐ but not histamine‐, A23187‐ or carbachol‐induced NO‐mediated relaxation. Vaspin significantly increased ACh‐induced eNOS phosphorylation and inhibited AChE activity. Conclusion We for the first time demonstrate that vaspin augments the ACh‐induced NO‐mediated endothelium‐dependent relaxation via the inhibition of AChE activity.