A method for production of a recombinant human alpha-Gal A comprising the steps of: (a)culturing recombinant human alpha-Gal A-producing mammalian cells in a serum-free medium to let them secrete the recombinant human alpha-Gal A in the medium, (b) collecting culture supernatant by removing the cells from the culture that is obtained in step (a) above, (c) subjecting the culture supernatant collected in step (b) above to anion-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions, (d) subjecting the fractions collected in step (c) above to hydrophobic column chromatography to collect recombinant human alpha-Gal A-active fractions, (e) subjecting the fractions collected in step (d) above to a column chromatography employing as solid phase a material having affinity for phosphate group to collect the recombinant human alpha-Gal A-active fractions, (f) subjecting the fractions collected in step (e) above to cation-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions, (g) subjecting the fractions collected in step (f) above to dye-affinity column chromatography to collect the recombinant human alpha-Gal A -active fractions, by using a dye affinity chromatography column equilibrated at pH 6.8-7.2 with phosphate buffer supplemented with 100-150 mM of sodium chloride and eluting the recombinant human alpha-GalA using a phosphate buffer containing 400-750 mM of sodium chloride and 40-60 mM of tris(hydroxymethyl)aminomethane, and (h) subjecting the fractions collected in step (g) above to gel filtration column chromatography to collect the recombinant human alpha-Gal A-active fractions, in the order. (a)culturing recombinant human alpha-Gal A-producing mammalian cells in a serum-free medium to let them secrete the recombinant human alpha-Gal A in the medium, (b) collecting culture supernatant by removing the cells from the culture that is obtained in step (a) above, (c) subjecting the culture supernatant collected in step (b) above to anion-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions, (d) subjecting the fractions collected in step (c) above to hydrophobic column chromatography to collect recombinant human alpha-Gal A-active fractions, (e) subjecting the fractions collected in step (d) above to a column chromatography employing as solid phase a material having affinity for phosphate group to collect the recombinant human alpha-Gal A-active fractions, (f) subjecting the fractions collected in step (e) above to cation-exchange column chromatography to collect the recombinant human alpha-Gal A -active fractions, (g) subjecting the fractions collected in step (f) above to dye-affinity column chromatography to collect the recombinant human alpha-Gal A -active fractions, by using a dye affinity chromatography column equilibrated at pH 6.8-7.2 with phosphate buffer supplemented with 100-150 mM of sodium chloride and eluting the recombinant human alpha-GalA using a phosphate buffer containing 400-750 mM of sodium chloride and 40-60 mM of tris(hydroxymethyl)aminomethane, and (h) subjecting the fractions collected in step (g) above to gel filtration column chromatography to collect the recombinant human alpha-Gal A-active fractions, in the order. <EOS>