ABSTRACT: Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the pathogenesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene expression was analyzed in human bronchoalveolar lavage fluid (BALF) cells and murine lung tissue after 5 h of ventilation. In addition, wild-type (WT) and TLR2 knockout (KO) mice were ventilated with either lower tidal volumes (VT) of 7 mL/kg with positive end-expiratory pressure (PEEP) or higher VT of 15 mL/kg without PEEP for 5 h. Spontaneously breathing mice served as controls. Total protein and immunoglobulin M levels in BALF, neutrophil influx into the alveolar compartment, and interleukin 6 (IL-6), IL-1β, and keratinocyte-derived chemokine concentrations in lung tissue homogenates were measured. We observed enhanced TLR2 gene expression in BALF cells of ventilated patients and in lung tissue of ventilated mice. In WT mice, ventilation with higher VT without PEEP resulted in lung injury and inflammation with higher immunoglobulin M levels, neutrophil influx, and levels of inflammatory mediators compared with controls. In TLR2 KO mice, neutrophil influx and IL-6, IL-1β, and keratinocyte-derived chemokine were enhanced by this ventilation strategy. Ventilation with lower VT with PEEP only increased neutrophil influx and was similar in WT and TLR2 KO mice. In summary, injurious ventilation enhances TLR2 expression in lungs. Toll-like receptor 2 deficiency does not protect lungs from ventilator-induced lung injury. In contrast, ventilation with higher VT without PEEP aggravates inflammation in TLR2 KO mice.