Ventilator-induced lung injury is associated with activation of inflammatory cytokines and excessive lung distention, which directly affects blood-gas barrier and lung vascular permeability. Small GTPases Rho and Rac play an essential role in agonist-mediated endothelial cell cytoskeletal remodeling and permeability, but their role in lung endothelial cell barrier regulation under mechanical stress is less understood. We studied magnitude-dependent effects of cyclic stretch on lung endothelial cell barrier restoration after thrombin challenge and linked these effects with differential activation of Rho and Rac. Consistent with differential effects of 5% cyclic stretch and 18% cyclic stretch on thrombin-induced endothelial cell monolayer disruption, a peak of thrombin-induced Rho activation at 5 minutes was enhanced by endothelial cell preconditioning at 18% cyclic stretch and was attenuated by 5% cyclic stretch. Endothelial cell preconditioning at physiological cyclic stretch (5%) induced more pronounced activation of Rac at 30 minutes compared to endothelial cell exposed to 18% cyclic stretch and caused nearly complete endothelial cell monolayer recovery observed after 50 minutes of thrombin stimulation, which was associated with peripheral accumulation of Rac effector cortactin. Promotion of endothelial cell barrier restoration by physiological cyclic stretch as well as cortactin peripheral accumulation was abolished by siRNA-based depletion of Rac expression or by Rac pharmacological inhibitor NSC-23766. Lung endothelial cell subjected to long-term cyclic stretch (18% elongation, 24 hours) increased expression of procontractile cellular proteins Rho, myosin light chain kinase, ZIP-kinase, and released extracellular bioactive molecules with barrier-disruptive properties. These results show involvement of Rac-mediated mechanisms in the barrier-promoting effects of physiologic cyclic stretch preconditioning and suggest direct link between amplitude-dependent activation of Rho- and Rac-mediated pathways and dynamic regulation of lung endothelial barrier by mechanochemical stimuli.