Agonist-induced activation of Rho GTPase signaling leads to endothelial cell (EC) permeability and may culminate in pulmonary edema, a devastating complication of acute lung injury. Cingulin is an adaptor protein first discovered in epithelium and is involved in the organization of the tight junctions. This study investigated the role of cingulin in control of agonist-induced lung EC permeability via interaction with RhoA-specific activator GEF-H1. The siRNA-induced cingulin knockdown augmented thrombin-induced EC permeability monitored by measurements of transendothelial electrical resistance and endothelial cell permeability for macromolecules. Increased thrombin-induced permeability in ECs with depleted cingulin was associated with increased activation of GEF-H1 and RhoA detected in pulldown activation assays. Increased GEF-H1 association with cingulin was essential for down-regulation of thrombin-induced RhoA barrier disruptive signaling. Using cingulin-truncated mutants, we determined that GEF-H1 interaction with the rod + tail domain of cingulin was required for inactivation of GEF-H1 and endothelial cell barrier preservation. The results demonstrate the role for association of GEF-H1 with cingulin as the mechanism of RhoA pathway inactivation and rescue of EC barrier after agonist challenge.