Vitronectin is present in large concentrations in serum and participates in regulation of humoral responses, including coagulation, fibrinolysis, and complement activation. Because alterations in coagulation and fibrinolysis are common in acute lung injury, we examined the role of vitronectin in LPS-induced pulmonary inflammation. Vitronectin concentrations were significantly increased in the lungs after LPS administration. Neutrophil numbers and proinflammatory cytokine levels, including IL-1β, MIP-2, KC, and IL-6, were significantly reduced in bronchoalveolar lavage fluid from vitronectin-deficient (vitronectin-/-) mice, as compared with vitronectin+/+ mice, after LPS exposure. Similarly, LPS induced increases in lung edema, myeloperoxidase-concentrations, and pulmonary proinflammatory cytokine concentrations were significantly lower in vitronectin-/- mice. Vitronectin-/- neutrophils demonstrated decreased KC-induced chemotaxis as compared with neutrophils from vitronectin+/+ mice, and incubation of vitronectin+/+ neutrophils with vitronectin was associated with increased chemotaxis. Vitronectin-/- neutrophils consistently produced more TNF-α, MIP-2, and IL-1β after LPS exposure than did vitronectin+/+ neutrophils and also showed greater degradation of IκB-α and increased LPS-induced nuclear accumulation of NF-κB compared with vitronectin+/+ neutrophils. These findings provide a novel vitronectin-dependent mechanism contributing to the development of acute lung injury. Copyright © 2007 by The American Association of Immunologists, Inc.