Abstract: Ulcerative colitis (UC) is a chronic relapsing inflammatory disease primarily affecting the colonic and rectal mucosa. Its complex pathogenesis, involving immune dysregulation, intestinal barrier dysfunction, gut microbiota dysbiosis, and other pathological factors, has led to the current lack of effective therapeutic agents in clinical practice. This study focused on extracting and characterizing polysaccharide components from the Rhubarb-Astragalus herb pair (GJ-2 and GJ-3) and evaluating their therapeutic effects on dextran sulfate sodium (DSS)-induced UC in mice. Crude polysaccharides were extracted from Rheum palmatum and Astragalus membranaceus via aqueous extraction-alcohol precipitation. Two polysaccharide fractions (GJ-2 and GJ-3) were isolated and purified using anion-exchange cellulose column. Structural characteristics, including total polysaccharide content (phenol-sulfuric acid method), uronic acid quantification (m-hydroxydiphenyl assay), infrared spectral features (FT-IR), and monosaccharide composition analysis 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization coupled with HPLC were systematically characterized. In vivo, a DSS-induced UC model was established using 2.5% dextran sulfate sodium (DSS). After intragastric administration of different doses of polysaccharide fractions, pathological changes of the colon tissue were observed by hematoxylin-eosin (HE) staining, while the levels of inflammatory factors in mouse serum and myeloperoxidase (MPO) activity in colon tissue were quantified by enzyme-linked immunosorbent assay (ELISA). Tight junction proteins (ZO-1, claudin-1) were analyzed via immunofluorescence, and tumor necrosis factor α (TNF-α)/interleukin 6 (IL-6) mRNA expression was quantified by qRT-PCR. Gut microbiota composition was evaluated through 16S rDNA sequencing of cecal contents. Consequently, two weakly acidic polysaccharide fractions, GJ-2 and GJ-3, were isolated from the Rheum palmatum-Astragalus membranaceus herb pair. Monosaccharide analysis revealed GJ-2 was composed of glucose (Glc), galactose (Gal), and arabinose (Ara), whereas GJ-3 predominantly contained Gal and Ara, both exhibiting carboxyl groups and pyranose configurations (FT-IR). In DSS-treated mice, GJ-2 and GJ-3 significantly ameliorated colon shortening, body weight loss, and elevated DAI scores. Histopathology demonstrated reduced mucosal damage, while ELISA and qRT-PCR showed decreased TNF-α and IL-6 levels in serum and colon tissues. Moreover, the polysaccharide fractions significantly upregulated the expression levels of ZO-1 and claudin-1 and restored gut microbiota diversity in DSS-induced mice, and enriched beneficial taxa, including f.__Clostridiales and g.__Vagococcus. In conclusion polysaccharide fractions from the Rheum palmatum-Astragalus membranaceus herb pair exhibit therapeutic potential against UC, likely serving as a key material basis for the efficacy of this herb pair. The animal operation was approved by the Zhejiang Center of Labortory Animals (No: ZJCLA-IACUC-20011023). All experiments were conducted in accordance with relevant guidelines and regulations.