Ulcerative colitis (UC), an idiopathic and recurrent ailment, substantially influences a patient's health. Mung bean peptides (MBPs) are bioactive substances derived from mung bean protein that possess notable anti-inflammatory properties. However, their efficacy and underlying mechanisms in UC treatment remain unclear. In this study, the structural characteristics of MBPs were examined by determining various parameters, such as amino acid composition, molecular weight distribution, and peptide sequences, thereby structurally demonstrating their anti-inflammatory potential. The therapeutic effectiveness of MBPs in UC treatment was evaluated by assessing its influence on colon length, histological damage to colonic tissue, and disease activity index of mice suffering from colitis induced by dextran sulfate sodium (DSS). Additionally, the study explored the potential mechanism of action of MBPs in UC by analyzing the intestinal microbiota, inflammatory cytokines in serum, and tight junction (TJ) proteins in the colon tissue of mice. The results revealed that MBPs significantly increased colon length, reduced colonic tissue damage, and decreased the disease activity index in mice with UC. MBPs restored intestinal barrier function by upregulating the expression of ZO-1 and claudin-1 proteins within the colonic tissue of mice with DSS-induced colitis, thereby treating UC. MBPs exerted anti-inflammatory effects by downregulating the amplification of inflammatory cytokines in the serum, improving the gut microbiota structure in mice with colitis, and regulating immune-related signaling pathways. Therefore, there is an experimental basis for the potential use of MBPs as adjunctive therapy in UC.