The colonic mucosal barrier is an important component of the intestinal barrier, and its integrity is crucial for maintaining digestive tract homeostasis and normal metabolism in the body. This study aimed to elucidate the mechanisms by which malvidin-3-O-galactoside (M3G) might ameliorate colonic mucosal barrier function, from the perspective of physical barrier function and immune barrier function. Male C57BL/6J mice were given dextran sulfate sodium (DSS) to establish a mice model for colitis and then administrated with or without M3G for one week. The results showed that M3G supplementation significantly improved the disease activity index (DAI) score and colon tissue injury in mice with DSS-induced colitis. M3G improved the colonic physical barrier function by modulating the expression of mucin2 (MUC2), claudin-1, occludin, zona occludens 1 (ZO-1), and intestinal fatty acid binding protein (iFABP) in the colonic mucosa. Additionally, M3G also relieved the colonic immune barrier of mice by increasing the level of secretory immunoglobulin A (SIgA) in colon tissue and the percentages of CD4+T (CD3+CD4+) and CD8+T (CD3+CD8+) cells in colon lamina propria monocytes in mice. Furthermore, M3G down-regulated Notch signaling pathway-related proteins such as Notch1, notch intracellular domain (NICD), delta-like ligand 4 (DLL4), delta-like ligand 1 (DLL1), and hairy/enhancer of split 1 (Hes1) of colon tissue. The present results demonstrated that M3G can improve colonic mucosal barrier function by inhibiting the Notch signaling pathway.