〔Abstract〕 Objective To study the effect of high mobility group box B1(HMGB1) gene knockout on alleviating acute lung injury and inhibiting toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) pathway of sepsis mice. Methods Wild-type(WT) mice were divided into WT-Sham group and WT-model group, and HMGB1 knockout(KO) mice were divided into KO-sham group and KO-model group. Sepsis ALI model was established by cecal ligation and perforation in WT-model group and KO-model group. Sham operation was performed in WT-Sham group and KO-Sham group. 24 h after modeling, the partial pressure of arterial oxygen(PaO2) was detected, oxygenation index(OI) was calculated, pathological changes of lung tissue were detected and lung injury score was calculated, the concentrations of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6), reactive oxygen species(ROS), malondialdehyde(MDA), superoxide dismutase(SOD), in serum and lung tissues and the expression of HMGB1, TLR4 and nuclear NF-κB in lung tissues were detected. Results The PaO2, OI and the concentration of SOD in serum and lung tissue of WT-model group were lower than those of WT-Sham group, the lung injury scores, the concentrations of TNF-α, IL-1β, IL-6, ROS and MDA in serum and lung tissue, and the expression levels of HMGB1, TLR4 and nuclear NF-κB in lung tissue were higher than those in WT-Sham group(P<0.05). HMGB1 was not expressed in lung tissue of KO-model group, and the concentrations of PaO2, OI and the concentration of SOD in serum and lung tissue of KO-model group were higher than those of WT-model group, the lung injury scores, the concentrations of TNF-α, IL-1β, IL-6, ROS and MDA in serum and lung tissue, and the expression levels of TLR4 and nuclear NF-κB in lung tissue were lower than those of the WT-model group(P<0.05). Conclusion HMGB1 gene knockout alleviates acute lung injury of sepsis mice, the related molecular mechanism may be the inhibition of TLR4/NF-κB pathway mediated inflammation and oxidative stress.