Metastatic and invasive potential is a barrier to the successful treatment of gastric cancer. N-acetylgluco-saminyltransferase V (GnT-V), a key enzyme catalyzing the formation of 1,6 N-acetylglucosamine (GlcNAc), has been demonstrated to display a distinct function in different types of tumors. The aim of this study was to investigate the role of GnT-V in the invasive potential of BGC823 human gastric cancer cells in vitro and the possible underlying mechanism. GnT-V was downregulated in BGC823 cells by oligo-siRNA transfection. Cell proliferation and invasiveness were assessed by CCK-8 assay, Tunel assay, scratch-wound assay as well as transwell assay. The products of GnT-V, β1-6 branching of asparagine-linked oligosaccharides, were determined by L-PHA lectin blot analysis. The expression of EGFRs, E-cadherin/vimentin and MMP-2/MMP-9 was analyzed both at the mRNA and protein levels. The results showed that downregulation of GnT-V decreased proliferation and the metastatic/invasive potential of BGC823 cells. The expression of EGFRs, E-cadherin/vimentin and MMP-9, molecules related to cancer metastasis and invasion in various tumors, were influenced correspondingly. These findings suggest that downregulation of GnT-V inhibited cell metastasis and invasion of BGC823 cells via EGFR signaling-initiated EMT phenotype and MMP-9 expression. These results provide a novel mechanism to explain the role of GnT-V in cell metastasis and invasion.