Ulcerative colitis (UC) is a non-specific inflammatory bowel disease with a high rate of recurrence, development of novel therapeutic approaches with high efficacy and few adverse effects are still needed. Dioscoreae persimilis is an edible plant that is widely consumed a remedy for gastrointestinal diseases in traditional Chinese medicine. Polysaccharides have been proven to have protective effects on UC. However, the role of polysaccharides from D. persimilis in UC have not been studied. The refined D. persimilis Polysaccharide (DP), which consists of glucose and galactose, was extracted and purified using three-phase partitioning (TPP) method. The primary chemical and structural characteristics of DP were investigated by UV, FT-IR, high-performance liquid chromatography (HPLC) and high-performance gel-permeation chromatography (HPGPC). Based on dextran sulfate sodium (DSS) induced UC in mice, the alleviatory effect of DP on UC was explored. DP was found to alleviate histopathological changes of colon, improve colonic antioxidant capacity and ameliorate inflammation response in colitis mice. Moreover, 16S rDNA sequencing of fecal revealed that DP could restore the diversity and composition of gut microbiota, especially up-regulates the abundance of Acetatifactor, Lachnospiraceae and Lactobacillus, and increase the ratio of Firmicutes/Bacteroidetes. This study suggests DP may be used as a promising nutritional supplement to ameliorate colitis.