Acute lung injury (ALI) is a devastating disorder of the lung by various causes and its cardinal features are tissue inflammation, pulmonary edema, low lung compliance, and widespread capillary leakage. Among phosphoinositide 3-kinases (PI3Ks), PI3K-γ isoform has been shown to play an important role in a number of immune/inflammatory responses. We investigated the role of PI3K-γ and its molecular basis in lipopolysaccharide (LPS)-induced ALI using a selective inhibitor for PI3K-γ, AS 605240 and LPS-treated C57BL/6 mice. Administration of AS 605240 to LPS-treated mice markedly reduced the pathophysiological features of ALI and the increased production of ROS, cytokines, adhesion molecule, and VEGF in the lung. Our results also showed that treatment of mice with LPS activates nuclear factor-kB (NF-kB) and degradation of inhibitory kBα (IkBα) through PI3K-γ. Additionally, infiltration of dendritic cells (DCs) and expression of toll-like receptor 4 (TLR4) were significantly increased in the lung of LPS-treated mice, and inhibition of PI3K-γ reduced the infiltration of DCs and TLR4 expression in the lung. These results indicate that PI3K-γ is critically involved in LPS-induced ALI by regulating IkBα/NF-kB pathway and innate immune responses. Based on our data we suggest that PI3K-γ isoform is a promising target for the treatment of ALI.