Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury (VILI) and pulmonary edema. Using pulmonary endothelial cells (EC) exposed to physiologically (5% CS) and pathologically (18% CS) relevant magnitudes of cyclic stretch, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with suppression of Rho-dependent signalling triggered by VEGF. 5% CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signalling, suggesting synergistic barrier-protective effects of physiologic CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signalling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS prior to HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiologic CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathologic CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of ALI/ARDS severity via small GTPase-dependent regulation of lung endothelial permeability.