Fucosylated glycoconjugates are involved in numerous biological events, and ␣-L-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in ␣-L-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into ␣-L-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima ␣-L-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (␤/␣) 8-like domain and a C-terminal ␤-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp 244 , and the Brønsted acid/base, Glu 266. Because T. maritima ␣-L-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.