As a chronic inflammatory disease, ulcerative colitis (UC) is relevant to a rising risk of colorectal cancer. Dihydroberberine (DHBB), a natural occurring isoquinoline alkaloid with various bioactivities, was found in many plants including Coptis chinensis Franch. (Ranunculaceae), Phellodendron chinense Schneid. (Rutaceae), and Chelidonium majus L. (Papaveraceae). However, its protective effect on UC is sparsely dissected out.To explore the protective role and underlying mechanism of DHBB on a model of colitis.Acute colitis model was established by gavage with 3% dextran sulfate sodium (DSS) for 8 days. Influence of DHBB on DSS-induced clinical symptoms and disease activity index (DAI) was monitored and analyzed. Pathological injury of colon tissues was examined by hematoxylin-eosin and Alcian blue staining. The expression of intestinal mucosal barrier function proteins, immune-inflammation related biomarkers and signal pathway key targets were determined by ELISA kit, Western blot, immunohistochemistry and qRT-PCR.DHBB treatment effectively alleviated DSS-induced UC by relieving clinical manifestations, DAI scores and pathological damage, which exerted similar beneficial effect to azathioprine (AZA), and better than berberine (BBR). In addition, DHBB significantly improved the gut barrier function through up-regulating the levels of tight junction proteins and mucins. Furthermore, DHBB dramatically ameliorated colonic immune-inflammation state, which was related to the decrease of colonic pro-inflammatory cytokines and immunoglobulin through blocking TLR4/MyD88/NF-κB signal pathway.These results demonstrated that DHBB exerted a significant protective effect on DSS-induced experimental UC, at least partly through suppressing immune-inflammatory response and maintaining gut barrier function.