Abstract Background Rac small GTPases play important roles in cytoskeleton and many cell functions including cell cycle, cell growth, cell adhesion and gene transcription. Here, we investigated the roles of Rac including Rac1 and Rac2 in lipopolysaccharide (LPS)-induced pulmonary injury. Methods After LPS was intratracheally instilled to lungs in mice, Rac, CDC42 and RhoA activation assay by pull-down and West blot, inflammatory cell infiltration assay by counting cell numbers and lung histological examination, pro-inflammatory mediator mRNA expression assay by quantitative RT-PCR, measurement of myeloperoxidase (MPO) activity, Evans Blue and albumin accumulation by spectrophotometry were performed to evaluate the roles of Rac in pulmonary injury by using its specific inhibitor, NSC23766. Results LPS challenge led to increases of both Rac1 and Rac2, but not CDC42 or RhoA activities in lungs, and intraperitoneal administration with NSC23766 inhibited both Rac1 and Rac2, but not CDC42 or RhoA activities. Treatment with NSC23766 at 1 or 3mg/kg not only reduced the inflammatory cells infiltration and MPO activities, but also inhibited pro-inflammatory mediators, tumor necrosis factor-α and interleukin-1β, mRNA expression. Moreover, in vitro neutrophil migration assay and in vivo microvascular permeability assay indicated that NSC23766 not only inhibited neutrophil transwell migration toward a chemoattractant, fMLP, but also reduced Evans Blue and albumin accumulation in LPS-challenged lungs. LPS activated both Rac1 and Rac2, but not CDC42 or RhoA activities in lungs, and specific inhibition of Rac activities by NSC23766 effectively alleviated LPS-induced injury. General significance Rac could be a potential target for therapeutic intervention of pulmonary inflammation.