Casticin, an active compound isolated from Vitex Fructus, has been reported to have anti-inflammatory effects in previous studies. The present study was designed to evaluate the protective effects of casticin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and clarify the underlying mechanisms. ALI was induced by intratracheal instillation of LPS. Casticin was administrated 1 h before or after LPS challenge. The results showed that casticin decreased LPS-induced lung histopathological changes, MPO activity, and lung wet/dry weight ratio. Casticin also inhibited the numbers of inflammatory cells and the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β production. Furthermore, casticin suppressed LPS-induced NF-κB activation, and NLRP3, ASC, and caspase-1 expression. In conclusion, the results suggested that casticin had a protective effect against LPS-induced ALI. Casticin protected against LPS-induced ALI by inhibiting inflammatory cytokines production through the inhibition of NF-κB and NLRP3 signaling pathways.