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Anoikis is an anchorage-dependent cell death in which an epithelial cell shows the apoptotic behaviors upon loss of adhesion with cells and extracellular matrix. Anoikis resistance is known to be a crucial step for cancer metastasis, and it is demanding that understanding of the mechanism underlying the process is required to control cancer metastasis at a molecular level. We have previously identified N- Acetylglucosaminyltransferase-V (GnT-V), a glycosyltransferase that catalyzes the addition of N- acetylglucosamine (GlcNAc) to the core moiety of N-glycan of glycoproteins, producing β1,6-linkages, is a key protein inducing metastasis in human colon cancer cells. And the secretion of galectin-3 binding protein (LGALS3BP) was lowered in GnT-V overexpressing WiDr cells. In this study, we hypothesized that GnT-V has an inhibitory effect on anoikis of cancer cells, while LGALS3BP stimulates the apoptotic process. To prove this, we established the stable transfectants (WiDr:GnT-V), the control cells (WiDr:mock). In addition, we investigated the molecular signatures in various colon cancer cell lines: WiDr, HCT116, SW480 and LoVo. To induce anoikis process, cells were cultured atop 1% soft agar-coated plate and their cellular and molecular responses were monitored. As a result, GnT-V was observed to mitigate anoikis and promote cell aggregation in soft- agar plate system. Among human colon cancer cells tested, HCT116 cells formed aggregates poorly and thus showed low cell viability. Of note, the secretion of LGALS3BP was higher than any other colon cancer cellline, which is in line with the result that anoikis resistance was observed in LGALS3BP-suppressed WiDr cells. Taken together, these results may suggest that GnT-V induces anoikis resistance of cancer cells either alone or in cooperation with the target molecules, such as LGALS3BP.