It is common knowledge that the crosstalk of gut microbiota (GM) and dendritic cells (DCs) are critical for the pathogenesis of inflammatory bowel disease (IBD). As a major bioactive constituent derived from the root of the Sophora flavescens, Oxymatrine (OMT) was used to treat IBD in China. However, it is still unknown whether OMT ameliorates IBD by regulating the crosstalk between DCs and GM. In the present study, after 10 days of OMT (100 mg/kg/day) treated mice with colitis induced by dextran sulfate sodium (DSS), the change rate of body weight, colon weight, colon weight index, colon length, DAI score and colonic pathological damage scores of colitis mice were significantly ameliorate, followed with fewer ulceration and inflammatory cell infiltration, the increased expression of IL-4 and IL-13, and the decreased expression of CCL-2, IL-33 and IFN-γ. The percents of inflammatory DCs (such as TNF-αDCs, iNOSDCs, CXCR5DCs and E-cadherinDCs) were markedly decreased, and the GM composition was regulated. Importantly, it is positive correlated between the efficacy of OMT on colitis, GM and inflammatory DCs. Meanwhile, Western blotting assay showed that OMT suppressed the activation of TLR4, Myd88, IRAK4, IRAK1, TRAF6, TAK1, TAB, MKK3, MKK6, P38, NF-κB in the TLR / NF-κB signaling pathway. In summary, OMT exhibits the protective effect against the DSS-induced experimental colitis, which was achieved by regulating the crosstalk of inflammatory DCs and GM, and inhibiting the TLR / NF-κB signaling pathway.