Estrogen has diverse effects on cardiovascular function, including regulation of the contractile response to vasoactive substances such as serotonin. The serotonin system recently emerged as an important player in the regulation of vascular tone in humans. However, hyperreactivity to serotonin appears to be a critical factor for the pathophysiology of hypertension. In this study, we examined the modulatory mechanisms of estrogen in serotonin-induced vasoconstriction by using a combinatory approach of isometric tension measurements, molecular biology, and patch-clamp techniques. 17β-Estradiol (E2) elicited a significant and concentration-dependent relaxation of serotonin-induced contraction in deendothelialized aortic strips isolated from male rats. E2 triggered a relaxation of serotonin-induced contraction even in the presence of tamoxifen, an estrogen receptor antagonist, suggesting that E2-induced changes are not mediated by estrogen receptor. Patch-clamp studies in rat arterial myocytes showed that E2 prevented Kv channel inhibition induced by serotonin. Serotonin increased Src activation in arterial smooth muscle required for contraction, which was significantly inhibited by E2. The estrogen receptor-independent inhibition of Src by E2 was confirmed in HEK293T cells that do not express estrogen receptor. Taken together, these results suggest that estrogen exerts vasodilatory effects on serotonin-precontracted arteries via Src, implying a critical role for estrogen in the prevention of vascular hyperreactivity to serotonin.