Beta1,6-acetylglucosaminyltransferase V (GnT-V) forms beta1,6 branching on the trimannosyl terminus of N-glycans, allowing for the production of beta1,6Glc-NAc-bearing oligosaccharides. These are used by healthy myeloid cells and cancer cells alike for systemic migration. GnT-V has multiple glycoprotein substrates and thereby exerts global effects on cancer progression, characteristic of a master regulator of metastasis. Yet little is known of the regulation of GnT-V expression by tumor cells. It was previously reported that fusion of macrophages with Cloudman S91 mouse melanoma cells produced macrophage-melanoma hybrids with up-regulated GnT-V expression regarding mRNA and enzymatic activity. Majority of these hybrids showed increased chemotactic motility in vitro and elevated metastatic potential in vivo. Here we attempted to understand this at the molecular genetic level focusing on DNA hypermethylation as a potentially key step. Treatment of cells with 5-Aza-dC, an inhibitor of DNA methylation, resulted in decreased expression of GnT-V mRNA and beta1,6-branched oligosaccharides along with reduced glycosylation of LAMP-1, a major substrate for GnT-V. This was accompanied by reduced chemotactic motility of the cells. The results suggested that DNA hypermethylation in some fashion stimulated GnT-V expression. We thus investigated the promoter region of the GnT-V gene for hypermethylation of CpG islands, comparing macrophage-melanoma hybrids of low and high metastatic potential with the parental melanoma cell line. Genomic DNA after bisulfite modification amplified from this region showed identical sequences between the cell lines. The findings indicated that differential methylation of the promoter region of GnT-V gene was not responsible for its transcriptional control, rather, appeared to be controlled through a negative regulator, nm23, whose own expression was regulated by hypermethylation. Although our studies involved a highly experimental system, the results further suggest that by whatever mechanism, reduction of GnT-V activity through 5-Aza-dC treatment might provide a new approach towards prevention of metastatic progression.