Objective-Estradiol (E2) mediates numerous beneficial effects assigned to estrogens, but whereas mechanisms have been described at the endothelial level, direct effects on vascular smooth muscle cells (VSMC) are poorly documented. As evidence accumulates regarding the role of RhoA in vascular pathophysiology and the benefit of RhoA-Rho associated protein kinase (Rock) pathway inhibition, we analyzed if E2 could inhibit it in VSMC. Methods and Results-We show that in VSMC, E2 inhibits the RhoA-Rock pathway in a time-and concentrationdependent manner. The inhibition of RhoA-Rock pathway results from E2-induced phosphorylation of the Ser188 of RhoA. Using pharmacological, transfection, and in vitro phosphorylation experiments, we demonstrate that AMP-activated protein kinase subunit alpha 1 (AMPK␣1) is activated by estrogen receptor stimulation and catalyzes RhoA phosphorylation induced by E2. Ex vivo, ovariectomy leads to an increase in the amplitude of phenylephrine-or serotonine-induced contractions of aortic rings in wild-type mice but not in AMPK␣1-knock-out mice or E2-supplemented animals. These functional effects were correlated with a reduced level of RhoA phosphorylation in the aorta of ovariectomized female, male, and AMPK␣1 knock-out mice. Conclusion-Our work thus defines AMPK␣1 as (1) a new kinase for RhoA and (2) Key Words: cell physiology Ⅲ g proteins Ⅲ signal transduction Ⅲ vasoconstriction Ⅲ estrogen I n the vasculature, RhoA and its downstream effector Rho associated protein kinase (Rock) have been shown to regulate processes such as vascular smooth muscle cell (VSMC) contraction, proliferation and differentiations, endothelial permeability, platelet activation, and leukocyte migration. 1 Accordingly, basal RhoA activity is required for homeostatic functions in physiological conditions, but its sustained overactivation has pathological consequences in the vascular system, particularly in VSMC. Activation of RhoAdependent pathways is involved in excessive contraction, and thereby increases blood pressure, but also in excessive cell growth and migration that participate in pathological cardiovascular remodeling. 2 Several regulatory proteins thus cooperate to provide a tight control of RhoA activity including RhoA exchange factors and RhoA GTPase activating proteins that control the GDP/GTP cycle, and Rho guanine dissociation inhibitor (RhoGDI) that sequesters RhoA in the cytosol. 3 In addition to this regulation and independently of GDP-GTP cycling, recent reports have proposed that phosphorylation/ dephosphorylation cycle also controls Rho protein activity. cAMP-and cyclic GMP-dependent protein kinases (PKA and PKG) phosphorylate RhoA on Ser188. 4,5 Both in vitro and in vivo experiments indicated that Ser188 phosphorylation of RhoA induces increased association to GDI leading to cytosolic accumulation of RhoA, 6 inhibition of RhoA-mediated target activation and functions 7 and vasodilation. 5 Recently, we showed that stimulation of angiotensin II type 2 receptor (AT 2 R) in VSMC induces RhoA ph...