Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by impaired intestinal mucosal barrier function, leading to persistent inflammation and tissue damage. Current therapies often fail to address barrier dysfunction, highlighting the need for innovative treatments. This study developed a novel therapeutic strategy by combining decellularized porcine small intestinal submucosa (D-SIS) with fibroblast growth factor 20 (FGF-20) to promote mucosal repair and restore barrier integrity in a TNBS-induced colitis rat model. The D-SIS-based hydrogel, supplemented with hyaluronic acid (HA), was designed to enhance FGF-20 stability and enable sustained drug release. Results showed that the FGF-20-loaded hydrogel (MAF) exhibited excellent rheological properties, erosion resistance, and controlled drug release, making it suitable for rectal administration. cell experiments demonstrated that MAF enhanced Caco-2 cell proliferation, migration, and tight junction protein expression, restoring epithelial barrier integrity. In the colitis model, MAF significantly reduced disease activity index (DAI) scores, attenuated inflammation, and restored mucosal morphology. Additionally, MAF promoted goblet cell regeneration, enhanced mucus secretion, and upregulated intestinal stem cell markers, indicating its ability to repair both epithelial and mucus barriers. In conclusion, the MAF hydrogel represents a promising therapeutic approach for UC by combining the regenerative properties of FGF-20 with the bioactive support of D-SIS.