Acute lung injury (ALI) frequently occurs after liver transplantation and major liver surgery. Proinflammatory mediators released by damaged liver after liver ischemia/reperfusion (I/R) injury might contribute to this form of ALI, but the underlying mechanisms have not been well characterized. High-mobility group box protein 1 (HMGB1), a recently identified proinflammatory cytokine, was found to be significantly higher in the serum after liver I/R injury. This study investigated whether HMGB1 was involved as a stimulating factor, and whether its downstream Toll-like receptor 4 (TLR4), p38 mitogen-activated protein kinase (p38MAPK), and activator protein-1 (AP-1) signaling pathways act as mediators in the development of liver I/R injury-induced ALI. Extensive ALI and lung inflammation was induced in a rat model of liver I/R injury. Serum HMGB1 was significantly higher after liver I/R injury, and more importantly, expression of HMGB1 mRNA and protein in the lung tissue was also significantly increased. We further found that liver I/R injury enhanced the expression of TLR4 mRNA and protein, and the activity of p38MAPK and AP-1 in the lung tissue. Inhibition of TLR4 expression in the lung tissue by infection with pGCSIL-GFP-lentivirus-expressing small hairpin RNAs targeting the TLR4 gene (TLR4-shRNA lentivirus) significantly attenuated ALI, lung inflammation, and activity of p38MAPK and AP-1 in the lung tissue. These findings indicate that HMGB1 might contribute to the underlying mechanism for liver I/R injury-induced ALI and that its downstream TLR4, p38MAPK, and AP-1 signaling pathways are potentially important mediators in the development of ALI.