Sex steroids control cell movement and tissue organization; however, little is known of the involved mechanisms. This report describes the ongoing dynamic regulation by estrogen of the actin cytoskeleton and cell movement in human vascular endothelial cells that depends on rapid activation of the actin-regulatory protein moesin. Moesin activation is triggered by the interaction of the C-terminal portion of cell membrane estrogen receptor α with the G protein Gα13, leading to activation of the small GTPase RhoA and of the downstream effector Rho-associated kinase. The resulting phosphorylation of moesin on Thr558 is the means of moesin's binding to actin and the remodeling of the actin cytoskeleton. This cascade of events ensues within minutes of estradiol administration and results in changes in cell morphology and to the development of specialized cell membrane structures such as ruffles and pseudopodia that are necessary for cell movement. These findings expand our knowledge of the basis of estrogen's effects on human cells, including the regulation of actin assembly, cell movement and migration. They highlight novel pathways of signal transduction of estrogen receptor α through nontranscriptional mechanisms. Furthermore, exposure of this estrogen receptor-dependent, nongenomic action of estrogen on human vascular endothelial cells is especially relevant to the present interest in the role of estrogen in cardiovascular protection.