We addressed the in vivo role of phosphatidylinositol 3-kinase-γ (PI3K-γ) in signaling the sequestration of polymorphonuclear leukocytes (PMNs) in lungs and in the mechanism of inflammatory lung vascular injury. We studied mice with deletion of the p110 catalytic subunit of PI3K-γ (PI3K-γ−/−mice). We measured lung tissue PMN sequestration, microvascular permeability, and edema formation after bacteremia induced by intraperitoneal Escherichia coli challenge. PMN infiltration into the lung interstitium in PI3K-γ−/−mice as assessed morphometrically was increased 100% over that in control mice within 1 h after bacterial challenge. PI3K-γ−/−mice also developed a greater increase in lung microvascular permeability after E. coli challenge, resulting in edema formation. The augmented lung tissue PMN sequestration in PI3K-γ−/−mice was associated with increased expression of the PMN adhesive proteins CD47 and β3-integrins. We observed increased association of CD47 and β3-integrins with the extracellular matrix protein vitronectin in lungs of PI3K-γ−/−mice after E. coli challenge. PMNs from these mice also showed increased β3-integrin expression and augmented β3-integrin-dependent PMN adhesion to vitronectin. These results point to a key role of PMN PI3K-γ in negatively regulating CD47 and β3-integrin expression in gram-negative sepsis. PI3K-γ activation in PMNs induced by E. coli may modulate the extent of lung tissue PMN sequestration secondary to CD47 and β3-integrin expression. Therefore, the level of PI3K-γ activation may be an important determinant of PMN-dependent lung vascular injury.