Hemorrhagic shock (HS) due to major trauma predisposes the host to the development of acute lung inflammation and injury. The lung vascular endothelium is an active organ that plays a central role in the development of acute lung injury (ALI) through generating reactive oxygen species (ROS), and synthesizing and releasing of a number of inflammatory mediators, including leukocyte adhesion molecules that regulate neutrophils emigration. Previous study from our laboratory has demonstrated that in a setting of sepsis, TLR4 signaling can induce TLR2 expression in endothelial cells (EC), and thereby increasing the cells' response to TLR2 ligands. The present study tested the hypothesis that TLR4 activation by HS and the resultant increased TLR2 surface expression in EC might contribute to the mechanism underlying HS-augmented activation of lung EC. The results show that HMGB1 through TLR4 signaling mediates HS-induced surface expression of TLR2 in the lung and mouse lung vascular endothelial cells (MLVEC). Furthermore, the results demonstrate that HMGB1 induces activation of NAD(P)H oxidase and expression of ICAM-1 in the lung and MLVEC sequentially depends on TLR4 in the early phase and on TLR2 in the late phase following HS. Finally, the data indicate an important role of the increased TLR2 surface expression in enhancing the activation of MLVEC and augmenting pulmonary neutrophil infiltration in response to TLR2 agonist peptidoglycan (PGN). Thus, induction of TLR2 surface expression in lung endothelial cells, induced by HS and mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for endothelial cell-mediated inflammation and organ injury following trauma and hemorrhage.