The Wnt/β-catenin signaling pathway and cadherin-mediated adhesion are implicated in epithelial-mesenchymal transition (EMT), a key cellular process in invasion and metastasis. Often, deregulation of the Wnt pathway is caused by altered phosphorylation of its components. Specifically, phosphorylation of Ser or Thr residues of β-catenin affects its location and interaction with E-cadherin, thus facilitates cell-cell adhesion. O-GlcNAcylation, the addition of O-GlcNAc to Ser or Thr, is comparable with phosphorylation and may compete with it on the same or adjacent residues. In this study, O-GlcNAcase, the enzyme responsible for the removal of O-GlcNAc, was inhibited. This inhibition resulted in global elevation of protein O-GlcNAcylation, increased expression of E-cadherin and b-catenin, and enhanced translocation of both proteins in fibroblasts and colon cancer cells. Moreover, concomitantly with O-GlcNAcylation elevation, fibroblast cell motility was enhanced. These results support the notion that O-GlcNAcylation affects EMT and cell motility, which may further influence metastasis and wound healing.