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Scope Ulcerative colitis (UC) is a severe disease of the intestinal tract. To investigate the role of TLR4/Mitogen‐activated protein kinase (MAPK)/Nuclear factor kappa‐B(NF‐κB) pathways and intestinal flora in UC, and the protective mechanisms of Mesona chinensis Benth polysaccharides (MBP), potential therapeutic agents due to their diabetes‐relieving, cancer‐suppressing, and immunomodulatory properties. Methods and results A dextran sulfate sodium (DSS)‐induced mouse colitis model is used for experiments; the histopathology, immunohistochemistry, and Western blotting’s results suggest that MBP can alleviate the colitis symptoms, inhibits the overproduction of TNF‐α, IL‐1β, promote IL‐10, reduces myeloperoxidase activity, and alleviates the inflammatory response probably by inhibiting the activation of TLR4/MAPK/NF‐κB pathways. Furthermore, MBP improvs the ratio of Bcl‐2/BAX, maintains the intestinal integrity by promoting the levels of zonulin occludin‐1 (ZO‐1), occluding and mucin mucin‐2 (MUC‐2), reduces the levels of endotoxin (ET), lipopolysaccharide binding protein (LBP) in serum, and oxidative stress in liver. Moreover, using 16S rRNA Gene Sequencing analysis, MBP regulates gut microbiota by decreasing the abundances of Helicobacter and Prevotella and increasing the abundances of Lactobacillus and Coprococcus, reverses microbiota dysbiosis caused by DSS. Conclusion These findings confirm the anti‐inflammatory effects of MBP, restoration of the intestinal barrier and intestinal flora, and have therapeutic potential to attenuate the development of UC.

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