The mammalian alpha 1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N-glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the alpha 1,3 mannose arm of N-glycan is essential for FUT8-catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin in a GnTI knock-out, FUT8-overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man(5)GlcNAc(2), suggesting that FUT8 can catalyze core fucosylation of N-glycans lacking an alpha 1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 by examining its in vitro activity toward an array of selected N-glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N-glycans lacking an unmasked alpha 1,3-arm GlcNAc moiety are not FUT8 substrates. However, Man(5)GlcNAc(2) glycan could be efficiently core-fucosylated by FUT8 in an appropriate protein/peptide context, such as with the erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary complex-type N-glycan also could be core-fucosylated by FUT8, albeit at much lower efficiency than the Man(5)GlcNAc(2) peptide. This study represents the first report of in vitro FUT8-catalyzed core fucosylation of N-glycans lacking the alpha 1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8.