RhoA, an important member of the Rho family of GTPases, has been implicated in many cellular processes. Our pilot study found that RhoA participated in the regulation of vascular reactivity after shock, but the mechanism was incompletely understood. Whether RhoA regulates vascular reactivity through the Rho kinase-myosin light-chain phosphatase (MLCP) and Rac1-p21-activated kinase (PAK)-myosin lightchain kinase (MLCK) signaling pathway needs investigation. With isolated, superior mesenteric arteries from hemorrhagic-shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), the effects of U-46619 (RhoA agonist) and C3 transferase (RhoA antagonist) on vascular reactivity, and the relationship to the Rho kinase-MLCP and Rac1-PAK-MLCK signaling pathways were observed. The vascular reactivity of the superior mesenteric artery and the contractile response of VSMCs to norepinephrine after prolonged hemorrhagic shock and hypoxia (2 h) were significantly decreased. Activation of RhoA with U-46619 significantly increased shock or hypoxia-induced decreased vascular reactivity. These effects of U-46619 were abolished by Y-27632 (Rho kinase inhibitor) and PDGF (Rac1 stimulator). Y-27632 had a stronger antagonistic effect than PDGF. U-46619 increased the activity of Rho kinase and MLCK, enhanced the phosphorylation of 20-kDa myosin light chain, and decreased the activity of Rac1, PAK, and MLCP in VSMCs after hypoxia. Y-27632-antagonized U-46619 induced the decrease of MLCP activity and the increase of 20-kDa myosin light chain phosphorylation. PDGF-antagonized U-46619 induced decrease of PAK activity and increase of MLCK activity. RhoA has an important role in the regulation of vascular reactivity after hemorrhagic shock. The Rho kinase-MLCP and Rac1-PAK-MLCK signal pathways participate in the regulatory process of RhoA. Rho kinase-MLCP may be the main signaling pathway by which RhoA regulates vascular reactivity. Copyright © 2010 the American Physiological Society.