This study investigated the biological activity of chestnut nonstarch polysaccharide (CNP) after removing starch. CNP was isolated from chestnut, with its monosaccharide composition identified as rhamnose, mannose, fructose, glucuronic acid, ribose, and galacturonic acid. Animal experiments showed that CNP can significantly alleviate the inflammatory response induced by dextran sodium sulfate (DSS) in a murine model of ulcerative colitis (UC). CNP alleviates colitis in mice by boosting antioxidant enzymes, reducing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, strengthening the intestinal barrier via tight junction proteins, and suppressing inflammation through the PI3K/NF-kappaB pathway. Results from 16S rDNA sequencing demonstrated that CNP intake significantly improved the richness and composition of the gut microbial community. These findings suggest that CNP exerts a protective effect against DSS-induced colitis by enhancing intestinal barrier integrity, mitigating oxidative stress, regulating cytokine levels, and restoring gut microbial balance. The results of this study highlight the important application value of CNP in the development of functional foods.