Background— Tissue edema and endothelial barrier dysfunction as observed in sepsis and acute lung injury carry high morbidity and mortality, but currently lack specific therapy. In a recent case report, we described fast resolution of pulmonary edema on treatment with the tyrosine kinase inhibitor imatinib through an unknown mechanism. Here, we explored the effect of imatinib on endothelial barrier dysfunction and edema formation. Methods and Results— We evaluated the effect of imatinib on endothelial barrier function in vitro and in vivo. In human macro- and microvascular endothelial monolayers, imatinib attenuated endothelial barrier dysfunction induced by thrombin and histamine. Small interfering RNA knock-downs of the imatinib-sensitive kinases revealed that imatinib attenuates endothelial barrier dysfunction via inhibition of Abl-related gene kinase (Arg/Abl2), a previously unknown mediator of endothelial barrier dysfunction. Indeed, Arg was activated by endothelial stimulation with thrombin, histamine, and vascular endothelial growth factor. Imatinib limited Arg-mediated endothelial barrier dysfunction by enhancing Rac1 activity and enforcing adhesion of endothelial cells to the extracellular matrix. Using mouse models of vascular leakage as proof-of-concept, we found that pretreatment with imatinib protected against vascular endothelial growth factor–induced vascular leakage in the skin, and effectively prevented edema formation in the lungs. In a murine model of sepsis, imatinib treatment (6 hours and 18 hours after induction of sepsis) attenuated vascular leakage in the kidneys and the lungs (24 hours after induction of sepsis). Conclusions— Thus, imatinib prevents endothelial barrier dysfunction and edema formation via inhibition of Arg. These findings identify imatinib as a promising approach to permeability edema and indicate Arg as novel target for edema treatment.