AIM: To explore the protective effect and mechanism of melittin against LPS induced acute lung injury. METHODS: Mice were intratracheally injected with LPS (5 mg/kg) to establish acute lung injury model. The lung coefficient of mice and the lung wet-dry mass ratio were determined. The lung tissue was stained with HE. The number of inflammatory cells and expression levels of related inflammatory factors in bronchoalveolar lavage fluid (BALF) were detected. The activities of serum SOD, MDA and GSH were detected. The expression of TLR4, NF-κB p65 and p-NF-κB p65 in lung tissues were determined by immunohistochemistry and Western blot. RESULTS: Melittin had a protective effect on LPS-induced ALI, as evidenced by significantly reduced lung coefficient, lung wet/dry mass ratio, the number of inflammatory cells and the expression level of inflammatory factors in BALF, and improved pulmonary pathology (P<0.05, P<0.01). Compared with model group, the serum MDA level of melittin group was significantly decreased (P<0.01), while the activities of SOD and GSH were significantly increased (P<0.05, P<0.01). In addition, melittin can significantly reduce the expression of TLR4, inhibit the phosphorylation of NF-κB p65, and reduce the inflammatory response (P<0.01). CONCLUSION: Melittin can significantly improve LPS-induced ALI, and the protective effect of melittin is mainly related to the inhibition of TLR4/NF-κB signaling pathway and the reduction of oxidative stress and inflammation.