BACKGROUND: Autoimmune hepatitis (AIH) is an immune-mediated liver disease of unknown etiology accompanied by intestinal dysbiosis and a damaged intestinal barrier. Berberine (BBR) is a traditional antibacterial medicine that has a variety of pharmacological properties. It has been reported that BBR alleviates AIH, but relevant mechanisms remain to be fully explored. METHODS: BBR was orally administered at doses of 100 mg⋅kg-1⋅d-1 for 7 days to mice before concanavalin A-induced AIH model establishment. Histopathological, immunohistochemical, immunofluorescence, western blotting, ELISA, 16S rRNA analysis, flow cytometry, real-time quantitative PCR, and fecal microbiota transplantation studies were performed to ascertain BBR effects and mechanisms in AIH mice. RESULTS: We found that liver necrosis and apoptosis were decreased upon BBR administration; the levels of serum transaminase, serum lipopolysaccharide, liver proinflammatory factors TNF-α, interferon-γ, IL-1β, and IL-17A, and the proportion of Th17 cells in spleen cells were all reduced, while the anti-inflammatory factor IL-10 and regulatory T cell proportions were increased. Moreover, BBR treatment increased beneficial and reduced harmful bacteria in the gut. BBR also strengthened ileal barrier function by increasing the expression of the tight junction proteins zonula occludens-1 and occludin, thereby blocking lipopolysaccharide translocation, preventing lipopolysaccharide/toll-like receptor 4 (TLR4)/ NF-κB pathway activation, and inhibiting inflammatory factor production in the liver. Fecal microbiota transplantation from BBR to model mice also showed that BBR potentially alleviated AIH by altering the gut microbiota. CONCLUSIONS: BBR alleviated concanavalin A-induced AIH by modulating the gut microbiota and related immune regulation. These results shed more light on potential BBR therapeutic strategies for AIH.