Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215–N225 of DAO and the corresponding region, R216–G220, of DDO. A DAO mutant, in which I215–N225 is substituted by R216–G220 of DDO, showed D-aspartate–oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215–N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220–Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220–Y224. All of the mutants exhibited decreased apparent Km values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, kcat app/Km app, for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.