Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal enzyme α-galactosidase (α-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human α-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (β/α)₈ domain with the active site and an antiparallel β domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of α-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in α-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human α-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.