Ulcerative colitis (UC) is an inflammatory bowel disease characterized by bloody diarrhea and colonic inflammation. Although Phaeodactylum tricornutum polysaccharides (PTPs) possess anti-inflammatory and immunostimulatory properties, the effects on colonic inflammation remain unclear. This study investigated the molecular structure of PTPs and their potential effects in dextran sulfate sodium (DSS)-induced colitis in mice. The molecular weight of PTPs was 12.56 kDa, and ribose, rhamnose, galactose, and glucose were the main monosaccharides. Treatment with PTPs significantly restored colitis symptoms, enhanced colonic antioxidant capacity (MPO, CAT, and SOD), and suppressed the overproduction of inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha). Additionally, PTPs markedly alleviated colonic pathological damage, repaired the intestinal barrier (MUC2, Claudin3, Occludin, and ZO-1), and promoted the expression of butyrate-producing genes. The abundance of Akkermansiaceae, Lactobacillaceae, and Muribaculum tended to increase, while that of Erysipelotrichaceae and Prevotellaceae tended to decrease. This modulation of the microbiota contributed to enhanced gut barrier integrity and reduced bacterial translocation, thereby promoting intestinal homeostasis and counteracting colitis progression. Collectively, PTPs ameliorated intestinal dysbiosis and immune dysfunction in colitis, supporting their potential as therapeutic agents for UC. These findings highlight the potential of PTPs as promising candidates for developing functional foods, supplements, or pharmacological interventions for colitis management.