Abstract: This study sought to explore the anti-ulcerative colitis (UC) effects of polysaccharides derived from sequential extraction—initial hot water extraction from Atractylodes macrocephala decoction pieces (yielding AMPQT), followed by alkaline extraction of the residue (yielding AMPJT)—and compare the efficacy of AMPJT to AMPQT. We quantified the polysaccharide content, uronic acid content, protein content, and molecular weight of both extracts. A mouse model of UC was induced using 2.5% dextran sodium sulfate (DSS). Mice were subsequently administered AMPQT, AMPJT, or mesalazine. Body weight, colon length, thymus index, and spleen index were measured to calculate the disease activity index (DAI). ELISA was employed to detect levels of TNF-α, IL-1β, IL-4, IL-6, IL-10, and malondialdehyde (MDA), as well as the activities of superoxide dismutase (SOD) and myeloperoxidase (MPO) in colon tissue. Pathological changes in colon tissues were evaluated via hematoxylin-eosin (HE) staining. Immunofluorescence was used to determine the expression levels of ZO-1, occludin, and MUC2, while serum FITC-dextran concentration was measured to assess intestinal permeability. Results demonstrated that compared with the model group, AMPJT significantly alleviated symptoms and pathological damage in UC mice, restored thymus (P<0.05) and spleen indices and colon length (P<0.01), reduced levels of TNF-α, IL-1β, IL-6, and MDA levels (P<0.01) along with MPO activity (P<0.05), and increased levels of IL-4 and IL-10 and SOD activity in colon tissue (P<0.01). Additionally, AMPJT significantly upregulated the expression of ZO-1 (P<0.05) and MUC2 (P<0.01) while reducing intestinal permeability (P<0.05). Compared with AMPQT, AMPJT exhibited significantly greater or superior efficacy in improving these indicators, with statistically significant differences particularly evident in restoring colon length, reducing IL-6 levels, and increasing IL-4 levels (P<0.05). AMPJT demonstrated significant anti-UC effects in mice, outperforming AMPQT.