Colitis is characterized by immune dysregulation and gut microbiota imbalance. This study investigates the therapeutic effects of polysaccharides from black mulberry (PBM) on dextran sulfate sodium-induced colitis in mice. PBM administration significantly alleviated colitis symptoms, including body weight loss, histological damage, and inflammation, while enhancing antioxidant capacity and strengthening tight junction protein expression. PBM modulated gut microbiota composition, notably increasing Weissella spp., which correlated with elevated short-chain fatty acids (SCFAs) and decreased pro-inflammatory markers. Colonic RNA sequencing revealed the role of PBM in attenuating colitis via the TNF-α/NF-κB/ICAM-1 signaling pathway. Fecal microbiota transplantation (FMT) from PBM-treated mice confirmed that microbiota modulation from PBM contributed to these therapeutic effects. These findings suggest PBM as a natural therapeutic agent for colitis, offering a multifaceted approach to restoring gut homeostasis through microbiota modulation and inflammatory pathway regulation. This study provides new insights into dietary polysaccharides as potential adjunctive therapies for colitis.