Protostemonine （PSN） is the main anti-inflammatory alkaloid extracted from the roots of Stemona sessilifolia （known as ＂Baibu＂ in traditional Chinese medicine）. Here, we reported the inhibitory effects of PSN on lipopolysaccharide （LPS）-induced macrophage activation in vitro and LPS-induced acute lung injury in mice. Macrophage cell line RAW264.7 cells and mouse bone marrow-derived macrophages （BMDMs） were treated with PSN （1, 3, 10, 30 and 100 pmol/L） for 0.5 h and then challenged with LPS （0.1 pg/mL） for 24 h. Pretreatment with PSN significantly inhibited LPS-induced phosphorylation of MAPKs and AKT, iNOS expression and NO production in the macrophages. C57BL/6 mice were intratracheally injected with LPS （5 mg/kg） to induce acute lung injury （ALl）. The mice were subsequently treated with PSN （10 mg/kg, ip） at 4 and 24 h after LPS challenge. PSN administration significantly attenuated LPS-induced inflammatory cell infiltration, reduced pro-inflammatory cytokine （TNF-α, IL-113 and IL-6） production and eliminated LPS-mediated lung edema. Furthermore, PSN administration significantly inhibited LPS-induced pulmonary MPO activity. Meanwhile, LPS-induced phosphorylation of p38 MAPK, iNOS expression and NO production in the lungs were also suppressed. The results demonstrate that PSN effectively attenuates LPS-induced inflammatory responses in vitro and in vivo; the beneficial effects are associated with the decreased phosphorylation of MAPK and AKT and the reduced expression of pro-inflammatory mediators, such as iNOS, NO and cytokines. These data suggest that PSN may be a potential therapeutic agent in the treatment of ALl.