A ctivation of latent TGF-β by the α_vβ₆ integrin is a critical step in the development of acute lung injury. However, the mechanism by which α_vβ₆-mediated TGF-β activation is regulated has not been identified. We show that thrombin, and other agonists of protease-activated receptor 1 (PAR1), activate TGF-β in an α_vβ₆ integrin–specific manner. This effect is PAR1 specific and is mediated by RhoA and Rho kinase. Intratracheal instillation of the PAR1-specific peptide TFLLRN increases lung edema during high-tidal-volume ventilation, and this effect is completely inhibited by a blocking antibody against the α_vβ₆ integrin. Instillation of TFLLRN during high-tidal-volume ventilation is associated with increased pulmonary TGF-β activation; however, this is not observed in Itgb6^–/– mice. Furthermore, Itgb6^–/– mice are also protected from ventilator-induced lung edema. We also demonstrate that pulmonary edema and TGF-β activity are similarly reduced in Par1^–/– mice following bleomycin-induced lung injury. These results suggest that PAR1-mediated enhancement of α_vβ₆-dependent TGF-β activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury, and they identify PAR1 and the α_vβ₆ integrin as potential therapeutic targets in this condition.