Silibinin, a natural product extracted from Silybum marianum (milk thistle), has been reported to have anti-inflammatory effect. The aim of this study was to explore the therapeutic effects and potential mechanisms of silibinin on lipopolysaccharide (LPS)-stimulated inflammatory responses in acute lung injury (ALI). Male BALB/c mice were conditioned with silibinin 1 h after intranasal instillation of LPS. After 12 h, the myeloperoxidase (MPO) level in lung tissues, the wet/dry (W/D) ratio, inflammatory cytokines in the bronchoalveolar lavage fluid (BALF), and histopathological examination of lung were detected. Our results showed that silibinin inhibited LPS-induced histopathological changes and MPO activity, as well as the wet/dry (W/D) ratio in the lung tissues. Furthermore, silibinin significantly inhibited LPS-induced inflammatory cytokines production in the BALF. In addition, silibinin suppressed LPS-induced NF-κB activation and the expression of NLRP3 inflammasome. These results indicate that silibinin exerts its anti-inflammatory effect by inhibiting NF-κB and NLRP3 signaling pathways.