Overproduction of reactive oxygen species (ROS) results in oxidative stress, which is known to damage the lung and consequently lead to various lung diseases. Airway epithelium is first cell layer to contact with microbes and air pollutants. In this study, we investigated the molecular basis of ROS generation induced by lipopolysaccharide (LPS) in A549 cells, an alveolar epithelial cell line. A549 cells or NHBE cells were stimulated with LPS. The ROS generation was measured in A549 cells or NHBE cells pre-treated with a selective inhibitor of phosphatidylinositol 3-kinase γ (PI3Kγ), AS 605240 or a ROS scavenger, pyridoxamine (PM). Treatment of A549 cells with LPS caused a significant increase of intracellular ROS generation. Pretreatment with the PI3Kγ inhibitor, AS 605240 decreased the LPS-induced increase of ROS generation, phosphorylation of Akt, and production of phosphatidyl 3,4,5-trisphosphate in A549 cells. Treatment of A549 cells with LPS also increased the nuclear factor-κB (NF-κB) in nucleus, accompanying an increase in phosphorylation of inhibitory κB-α, degradation of the protein, and reduction of cytosolic NF-κB. Pretreatment with AS 605240 reduced the LPS-induced these changes. In addition, pretreatment with PM resulted in inhibition of nuclear NF-κB activation. Moreover, exogenous oxygen radical, hydrogen peroxide induced the activation of NF-κB in A549 cells. These results suggest that PI3Kγ plays a key role in LPS-induced ROS generation in alveolar epithelial cells therewith activating NF-κB.