To economically produce recombinant human α-galactosidase A (GLA) with a cell culture system that does not require bovine serum, we chose methylotrophic yeast cells with the OCH1 gene, which encodes α-1,6-mannosyltransferase, deleted and over-expressing the Mnn4p (MNN4) gene, which encodes a positive regulator of mannosylphosphate transferase, as a host cell line. The enzyme (yr-hGLA) produced with the gene-manipulated yeast cells has almost the same enzymological parameters as those of the recombinant human GLA produced with cultured human fibroblasts (agalsidase alfa), which is currently used for enzyme replacement therapy for Fabry disease. However, the basic structures of their sugar chains are quite different. yr-hGLA has a high content of phosphorylated N-glycans and is well incorporated into the kidneys, the main target organ in Fabry disease, where it cleaves the accumulated glycosphingolipids. A glycoprotein production system involving this gene-manipulated yeast cell line will be useful for the development of a new enzyme replacement therapy for Fabry disease.