Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC) are chronic recurrent nonspecific intestinal disease. Current IBD therapeutics cannot fundamentally change the natural course of IBD. Therefore, it is of great significance to find new treatment strategies for IBD. Preclinical and clinical studies have shown that mesenchymal stem cells (MSCs) are a promising therapeutic approach. However, the mechanism by which MSCs alleviate colitis and how MSCs affect intestinal mucosal barrier is still unclear. LPS-exposed human colonic epithelial cancer cell lines Caco2 and HT29. Dextran sulfate sodium (DSS)-induced IBD mouse were treated with MenSCs. We found that LPS downregulates intercellular junction proteins and induces the production of inflammatory cytokines in intestinal epithelial cells. MenSCs reduced paracellular permeability and restored barrier integrity in Caco2 cells. In Vivo, MenSCs mitigated DSS-induced colitis in mice by reducing body weight loss, colonic shortening, and disease activity index scores and by inhibiting the expressions of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. MenSCs increased the expression of TJ proteins, improved the destruction of tight junction (TJ) structures, and reduced intestinal epithelial permeability. Furthermore, MenSCs could inhibit NF-κB p65 phosphorylation and the expression of Snail and prevent Snail nuclear localization, thereby maintaining tight and adherens junctions. Our findings demonstrate that MenSCs alleviate intestinal inflammation and enhance barrier function by suppressing the NF-κB/Snail signaling axis, offering a promising therapeutic strategy for inflammatory bowel diseases.