Glehniae Radix has a wide range of pharmaceutical applications, and research on its main components has mainly focused on coumarins, alkaloids, lignans, and flavonoids, while neglecting the research on polysaccharides. Literature reports and our previous studies have shown that polysaccharides have certain therapeutic significance in immune regulation, antioxidant, anti-inflammatory and other aspects. Herein, the rat model of ulcerative colitis (UC) was established to evaluate the anti-inflammatory efficacy of the prepared Glehniae Radix polysaccharide (GLP) from the perspectives of inflammatory factors, intestinal tissue morphology, and microflora changes. The polysaccharides are mainly composed of galacturonic acid, rhamnose, glucose, galactose, and arabinose in molar ratios of 1.4:9.2:33.3:2.5:2.9, and GLP could downregulate the expression pro-inflammatory factors (interleukin 6, tumor necrosis factor α, and interferon γ) and significantly upregulate the expression of anti-inflammatory factor (interleukin 10). In addition, Glehniae Radix aqueous extract (GLA), GLP with low dosage and GLP with high dosage (GLPH) could increase the number of goblet cells, enhance the integrity of crypt structure, and reverse the status of inflammatory infiltrating cells. Moreover, GLA and GLPH could upregulate Lactobacillus and Lachnoclostridium in UC rats, and appropriately downregulate Lachnospiraceae_NK4A136_group, thereby optimizing the proportion of bacterial flora and improving the intestinal microbial environment. Our findings not only be valuable as theoretical materials for the further clinical applications of GLP, but the identified biomarkers and metabolic pathways also provide new clues for the diagnosis of UC.