The infiltration of polymorphonuclear neutrophils (PMNs) into lungs is an important feature of ventilator-induced lung injury (VILI) associated with pneumonia, but the mechanisms that recruit PMNs are poorly understood. Using Toll-like receptor 4 knockout mice (TLR4−/−), we addressed the role of TLR4 signaling in PMN recruitment. Wild type (WT) or TLR4−/− mice were challenged by intratracheal instillation of LPS (3.0 mg/kg) for 2 hr and then subjected to high tidal volume mechanical ventilation (28 ml/kg) for 2 hr. Ventilated WT mice exhibited significant increases in PMN sequestration (5-fold), bronchoalveolar lavage PMN count (3.6-fold), airway fluid albumin concentration (2.4-fold), and edema formation (1.6-fold). Quantitative lung histopathology demonstrated PMN infiltration, alveolar hemorrhage, edema, and alveolar wall thickening in lungs of ventilated, LPS-challenged WT mice. However, TLR4−/− mice showed negligible PMN sequestration, microvascular barrier breakdown, and edema formation. Thus, high tidal volume ventilation during pneumonia induces PMN sequestration and lung injury via TLR4-dependent signaling pathways. The results suggest the important role of lung innate immunity regulated by TLRs in the mechanism of PMN sequestration and injury resulting from mechanical ventilation at high tidal volumes. Supported by NIH R01 HL 071626, P01 HL 60678, and P01 HL 077806.