N-acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes β1-6 branching of N-acetylglucosamine on asparagine (N)-linked oligosaccharides (N-glycan) of cell proteins and the dysfunction of which is a common feature of various carcinomas. Nevertheless, the role of GnT-V remains controversial. Therefore, the clinical implication of GnT-V expression may differ in each cancer type. The implication of GnT-V status in patients with gastric cancer has not been studied extensively. In the present study, we examined GnT-V expression in gastric cancer specimen both at protein and mRNA levels. We compared GnT-V expression with clinical and pathologic variables. Kaplan-Meier survival curves were generated to show the cause-specific survival. Furthermore, the small interfering RNA was devised to downregulate the GnT-V mRNA expression in SGC7901 and BGC 823 cells. We characterized the function implication of GnT-V by cell proliferation and invasiveness analysis. Analysis in gastric cancer specimen revealed that GnT-V expression correlated with tumor grade and stage. The overall survival time of positive GnT-V expression in gastric cancer was significantly shorter than that of negative GnT-V expression. Moreover, the downregulation of GnT-V expression by small interfering RNA resulted in a decrease of cell proliferation and invasiveness in SGC7901 and BGC 823 cells accompanied by morphological change. This supports that GnT-V correlates with metastasis and prognosis in gastric cancer. These results contribute to new insight into the underlying molecular mechanisms of GnT-V regulation in gastric cancer with potential translational clinical applications.