β-Sitosterol (24-ethyl-5-cholestene-3-ol) is a common phytosterol Chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. In this study we investigated the effects of β-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. We demonstrate that β-sitosterol (150-450 μg/mL) dose-dependently suppresses inflammatory response through NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling in influenza A virus (IAV)-infected cells, which was accompanied by decreased induction of interferons (IFNs) (including Type I and III IFN). Furthermore, we revealed that the anti-inflammatory effect of β-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene I (RIG-I) signaling, led to decreased STAT1 signaling, thus affecting the transcriptional activity of ISGF3 (interferon-stimulated gene factor 3) complexes and resulting in abrogation of the IAV-induced proinflammatory amplification effect in IFN-sensitized cells. Moreover, β-sitosterol treatment attenuated RIG-I-mediated apoptotic injury of alveolar epithelial cells (AEC) via downregulation of pro-apoptotic factors. In a mouse model of influenza, pre-administration of β-sitosterol (50, 200 mg·kg·d, i.g., for 2 days) dose-dependently ameliorated IAV-mediated recruitment of pathogenic cytotoxic T cells and immune dysregulation. In addition, pre-administration of β-sitosterol protected mice from lethal IAV infection. Our data suggest that β-sitosterol blocks the immune response mediated by RIG-I signaling and deleterious IFN production, providing a potential benefit for the treatment of influenza.