Chitosan oligosaccharides (COS) have been shown to regulate various cellular and biological functions. The aim of this study was to investigate the antimetastatic potency of COS and the underlying mechanism. Here, we established a stably N-acetylglucosaminyltransferase V (GnT-V)-overexpressed MCF10A cell line. As expected, GnT-V overexpression greatly promoted cell migration in the transfectants by using wound healing assay. However, the induction in the cell migration was significantly suppressed by an addition of COS. Curiously, COS inhibited the protein expression of GnT-V in a dose dependent manner. Consistent with that, the reactivities with datura stramonium (DSA) and leuko-agglutinating phytohemagglutinin (L4-PHA) lectins, which specifically recognize branched N-acetylglucosamine (GlcNAc) structure, were also suppressed by COS. Taken together, these results demonstrated COS inhibited breast epithelial cell migration through down-regulation of GnT-V and its products, branched N-glycans, indicating that COS may serve as a potential novel therapeutic candidate for the treatment of metastatic breast cancer.