Steamed Polygonatum sibiricum is widely applied in clinical practice for its tonic effect on gastrointestinal tract. A novel polysaccharide named PSSP-EF was extracted from the steamed roots of Polygonatum sibiricum using hot water extraction, ethanol precipitation, and chromatographic purification. PSSP-EF, with a molecular weight of 2.24 x 10(4) Da, was consisted of mannose, glucosamine hydrochloride, glucose, galactose, xylose, and arabinose in a molar ratio of 10.25: 0.26: 3.56: 80.55: 1.72: 3.66, and its main chain was constituted by-->4)-beta-D-Galp-(1-->4)-beta-D-Galp-(1--> and -->4)-beta-D-Manp-(1-->4)-beta-D-2ace-Manp-(1--> residues, with branching from beta-D-Galp-(1--> residues. PSSP-EF could dramatically relieve clinical symptoms of ulcerative colitis (UC) in mice. Treatment with PSSP-EF significantly alleviated colon inflammation (TNF-alpha, IL-1beta, and IL-6), repaired intestinal mucosal barrier (Occludin, ZO-1, and Claudin-1) and regulated the balance of gut microbiota by increased the levels of Muribaculaeae, while decreasing the levels of Bacteroides, Erysipelatoclostridium, and Romboutsia. Notably, PSSP-EF remarkably increased the levels of acetic, propionic, isobutyric, butyric, valeric, and isovaleric acid in the cecal contents of UC mice. In conclusion, PSSP-EF has a significant therapeutic effect on UC by balancing gut microbiota, protecting intestinal mucosal barrier, and regulating short-chain fatty acid production, and can be developed as a functional food.