Glycosylation drives critical processes important for mammalian cell-cell and cell-matrix interactions. Alpha-l-fucose (α-l-f) is a key monosaccharide component of oligosaccharides that has been found to be overexpressed during tumor progression. Modification of cell surface fucosylation, we hypothesized, alters tumor cell phenotype and function at the end of the neoplastic progression cascade including tumor invasion. Alpha-l-fucosidase (α-l-fase) is a glycosidase that specifically removes (α-l-f) from oligosaccharide sites. We first verified the effectiveness of the α-l-fase to specifically decrease the level of α-l-f on the cell surface of several human breast cancer cell lines and also examined the recovery time for these cells to repopulate their surfaces. To investigate the potential effect of defucosylation on tumor functions, we studied the proliferation, and invasion in vitro of human breast cancer MDA-MB-231 cells as the representative cell model. We further examined several fucose-associated molecules previously shown to be involved in tumor progression, including CD44 and CD15 (Lewis X antigen). We found that α-l-fase pretreatment significantly decreased the invasive capability of breast cancer cells. Deoxyfuconojirimycin (DFJ), a specific α-l-fase inhibitor, reversed this effect. After fucosidase treatment, the level of both CD15 and CD44 were found to be reduced as measured by flow cytometry. α-l-fase treatment, further, did not affect tumor cell proliferation in vitro under identical experimental conditions. Gelatin zymography of conditioned media from tumor cells treated with α-l-fase demonstrated no change in MMP-2 activity while MMP-9 was significantly reduced. In summary, fucose containing glycans were found widely distributed on the cell surface of breast cancer cells and could be effectively removed by α-l-fase treatment. This decreased fucosylation, in turn, was seen to impair the interaction between tumor cells and extracellular matrices, and thus affected key cell functions modulating tumor invasion. Further elucidation of the molecular pathways involved in the inhibition of tumor cell invasion may suggest a rationale for the use of glycobiologic therapeutics to deter tumor progression. © 2008 Arányi Lajos Foundation.