Disclosed is a method for production of recombinant human alpha-galactosidase A (rh alpha-Gal A) in a large scale, with a high purity. The method comprises the steps of (a) culturing rh alpha-Gal A-producing mammalian cells in a serum-free medium, (b) collecting culture supernatant, (c) subjecting the culture supernatant to anion-exchange column chromatography, (d) to hydrophobic column chromatography, (e) to a column chromatography employing as solid phase a material having affinity for phosphate group, (f) to cation-exchange column chromatography, (g) to dye-affinity column chromatography, and (h) to gel filtration column chromatography, in the order.
