Background/Objectives: Chronic colitis presents a growing global health burden with rising incidence. This study investigated the ameliorative effect of Dendrobium officinale polysaccharide (DOP) against dextran sulfate sodium (DSS)-induced chronic colitis in mice, specifically examining its dual modulation of gut microbiota and metabolic pathways. Methods: DOP was extracted and purified from Dendrobium officinale stems and leaves. A chronic colitis model was established in male C57BL/6J mice via DSS induction. Eighty-four mice were randomized into seven groups: control, model, low/high-dose leaf-DOP, low/high-dose stem-DOP, and sulfasalazine positive control. We assessed body weight, disease activity index (DAI), colon length, splenic/thymic indices, inflammatory cytokines, and histopathology (Hematoxylin and Eosin/Alcian blue staining), with tight junction protein and tumor necrosis factor-alpha (TNF-α) expression quantified via immunofluorescence. 16S rRNA sequencing and untargeted metabolomics evaluated microbial and metabolic shifts. Results: DOP significantly attenuated colitis severity, restored colon histoarchitecture, elevated goblet cell counts, upregulated zonula occludens-1 (ZO-1) and occludin expression, and suppressed TNF-α. Crucially, DOP remodeled dysbiosis by enriching beneficial taxa (e.g., Candidatus_Saccharimonas, Lachnoclostridium) while reducing pathogens (Mucispirillum). Metabolomics further elucidated DOP-mediated regulation of purine and nicotinate/nicotinamide metabolism—pathways mechanistically linked to its anti-inflammatory and barrier-repair effects. Conclusions: DOP effectively alleviates symptoms of DSS-induced chronic colitis in mice, protects intestinal barrier integrity, and achieves therapeutic potential through simultaneous regulation of the gut microbiome and metabolome.

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