N-Glycans are involved in numerous biologic processes, such as cell adhesion, migration, and invasion. To distinguish the functions of complex high-mannose types of N-glycans, we used the clustered, regularly interspaced, short palindromic repeats/Cas9 system to establish N-acetylglucosaminyltransferase (GnT)-I-knockout (KO) cells. Loss of GnT-I greatly induced cell-cell adhesion and decreased cell migration. In addition, the expression levels of epithelial-mesenchymal transition (EMT) markers such as α-SMA, vimentin, and N-cadherin were suppressed, whereas the expression of claudin-1 was promoted, suggesting a mesenchymal-epithelial transition-like phenotype, an opposite process to the EMT, was occurred in the KO cells. The phosphorylation levels of Smad-2, epidermal growth factor receptor, and integrin-mediated focal adhesion kinase (FAK) were consistently suppressed. Furthermore, the restoration of GnT-I in the KO cells suppressed the cell-cell adhesion and augmented the expression of EMT markers as well as that of FAK activation. The expression levels of integrins were upregulated in the KO cells, although their functions were decreased, whereas their expression levels were downregulated in the rescued cells, which suggests a negative feedback loop between function and expression. Finally, we also found that the expression of GnT-I was important for cell survival, resistance to cancer drugs, and increased colony formation. The results of the present study demonstrate that GnT-I works as a switch to turn on/off EMT, which further supports the notion that on most surface receptors, the N-glycans differentially play essential roles in biologic functions.-Zhang, G., Isaji, T., Xu, Z., Lu, X., Fukuda, T., Gu, J. N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.