Fucosylated glycoconjugates have critical roles in biological processes, but a limited availability of α-l-fucosidase has hampered research on this human enzyme (h-Fuc) at a molecular level. After overexpressing h-Fuc in Escherichia coli as an active form, we investigated the catalytic function of this recombinant enzyme. Based on sequence alignment and structural analysis of close homologues of h-Fuc, nine residues of glutamate and aspartate in h-Fuc were selected for mutagenic tests to determine the essential residues. Among the mutants, D225N, E289Q, and E289G lost catalytic activity significantly; their kcat values are 1/5700, 1/430, and 1/340, respectively, of that of the wild-type enzyme. The Brønsted plot for kcat/Km for the E289G mutant is linear with β1g = -0.93, but that for kcat is biphasic, with β1g for poor substrates being -0.88 and for activated substrates being -0.11. The small magnitude of β1g for the activated substrates may indicate that the rate-limiting step of the reaction is defucosylation, whereas the large magnitude of the latter β1g value for the poor substrates indicates that the rate-limiting step of the reaction becomes fucosylation. The kinetic outcomes support an argument that Asp225 functions as a nucleophile and Glu289 as a general acid/base catalyst. As further evidence, azide significantly reactivated D225G and E289G, and 1H NMR spectral analysis confirmed the formation of β-fucosyl azide and α-fucosyl azide in the azide rescues of D225G and E289G catalyses, respectively. As direct evidence to prove the function of Glu289, an accumulation of fucosyl-enzyme intermediate was detected directly through ESI/ MS analysis.