Isoorientin (Iso) is a natural flavonoid present in the human diet, known for its anti-inflammatory, antioxidant, and antiviral properties. The purpose of this research was to illustrate the anti-inflammatory potential of Iso in ulcerative colitis (UC) mice and elucidate potential mechanisms. The mice model of UC was induced by cyclic intervention with Dextran Sulfate Sodium (DSS). We evaluated the efficacy of Iso in the treatment of UC, detected the expression of proteins related to the Galectin-3/NLRP3/IL-1β signaling pathway, and assessed the intestinal mucosa barrier function of colon tissue. Our findings demonstrated that Iso could improve colon length, the index of colonic weight, pathological damage to the colon, and serum cytokine secretion of UC mice. Most importantly, Iso could competitively bind Galectin-3, inhibit the interaction of Galectin-3 with NLRP3 and the expression of Galectin-3, NLRP3, ASC, caspase-1, IL-1β, and IL-18, increase the expression of MUC2, Occludin, and ZO-1. In summary, Iso had a therapeutic effect on UC mice, and its mechanism may be to inhibit the inflammatory response and improve the intestinal mucosa barrier by binding Galectin-3 and inhibiting the Galectin-3/NLRP3/IL-1β signaling pathway.