Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI: attenuation of LPS-induced lung injury, but exacerbation of ventilator-induced lung injury (VILI). Due to the critical role of MV in the care of ARDS patients, in the current study we pursued an assessment of the effectiveness of imatinib in a "two-hit" model of ALI caused by combined LPS and VILI. Imatinib significantly decreased BAL protein, total cells, neutrophils, and TNFα levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically-relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 hours after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NFB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.