Abstract: The paper aims to investigate the mechanism and protective effects of β-sitosterol on mice with Lipopolysaccharide (LPS)-induced acute lung injury (ALI). We evaluated the effect of β-sitosterol on LPS-induced production of TNF-α and interleukin (IL)-6 in the culture supernatants of RAW 264.7 cells by enzyme-linked immunosorbent assay (ELISA). Model of ALI in BALB/c mice was built by LPS (0.5 mg/kg) intranasal instillation and the severity of pulmonary injury was evaluated 24 h after LPS challenge. The inflammatory cytokines were assayed with ELISA in bronchoalveolar lavage fluid (BALF); the ratio of lung wet weight to dry weight (W/D) and water content were also measured; lung tissues were stained with Hematoxylineosin to observe the pathological. In addition，the activity changes of nuclear factor-κB (NF-κB) were detected by western blot. β-sitosterol could dose-dependently decrease the levels of TNF-α and IL-6 in vitro and in vivo. Pretreatment with β-sitosterol was found to decrease the W/D ratio and water content of the lung tissue and suppress lung edema and pulmonary alveolar damage at the same time. In addition, β-sitosterol suppressed not only the phosphorylation of NF-κB but also the degradation of its inhibitor (IκBα). β-sitosterol had good protective effect against ALI induced by LPS, and its mechanism might be related with down-regulated inflammatory cytokines (TNF-α, IL-6) and NF-κB expressions.