Gastric cancer is the second leading cause of cancer deaths worldwide, and more understanding of its molecular basis is urgently needed. Gastric gland mucin secreted from pyloric gland cells, mucous neck cells, and cardiac gland cells of the gastric mucosa harbors unique O-glycans carrying terminal α1,4-linked N-acetylglucosamine (αGlcNAc) residues. We previously reported that αGlcNAc loss correlated positively with poor outcomes for patients with differentiated-type gastric cancer. However, the molecular mechanisms underlying these outcomes remained poorly understood. Here, we examined the effects of upregulated αGlcNAc expression on malignant phenotypes of the differentiated-type gastric cancer cell lines, AGS and MKN7. Upregulation of αGlcNAc following ectopic expression of its biosynthetic enzyme attenuated cell proliferation, motility, and invasiveness of AGS and MKN7 cells in vitro. Moreover, AGS cell tumorigenicity was significantly suppressed by αGlcNAc overexpression in a xenograft model.To define the molecular mechanisms underlying these phenotypes, we investigated αGlcNAc binding proteins in AGS cells and identified Mucin-1 (MUC1) and podocalyxin.Both proteins were colocalized with αGlcNAc on human gastric cancer cells. We also found that αGlcNAc was bound to MUC1 in murine normal gastric mucosa. When we assessed the effects of αGlcNAc binding to MUC1, we found that αGlcNAc blocked galectin-3 binding to MUC1, phosphorylation of the MUC1 C-terminus, and recruitment of Src and β-catenin to that C-terminus. These results suggest that αGlcNAc regulates cancer cell phenotypes by dampening MUC1 signal transduction. K E Y W O R D S differentiated-type gastric cancer, invasion, malignant phenotype, tumor suppressor, αGlcNAcbinding protein 1 | INTRODUC TI ON Malignant neoplasms are leading causes of death in every country in the world and are important factors in decreasing life expectancy. 1 According to WHO estimates for 2019, malignant neoplasms are the leading or second highest cause of death before the age of 70 in 112 of 183 countries, and rank as third or fourth in another 23 countries. 2 Among them, gastric cancer is the second leading cause of