Inflammatory Bowel Disease (IBD) is a chronic autoimmune disorder that severely affects the gastrointestinal tract and is difficult to cure. This study explored the mechanism by which human umbilical cord mesenchymal stem cell-derived exosomes (HucMSC-Ex) alleviate IBD through O-GlcNAc glycosylation modification and the expression of related proteins. The study analyzed the effects of HucMSC-Ex on the inhibition of pro-inflammatory factors and promotion of intestinal epithelial cells regeneration in vitro and in vivo, with a focus on the role of the O-GlcNAc glycosylation of the RACK1 protein. The findings indicated that HucMSC-Ex reverses epithelial-mesenchymal transition (EMT) by upregulating O-GlcNAc glycosylation levels and effectively alleviates IBD symptoms and inflammatory responses in mouse intestinal epithelial cells. By modulating O-GlcNAc glycosylation, HucMSC-Ex exhibits significant therapeutic potential in immune regulation and gut microbiota remodeling, offering new perspectives for IBD treatment.