Two fungal α-glucosidases from Podospora anserina (PAG) and Schwanniomyces occidentalis (SOG) have different regioselectivity thought their high similarity in amino acid sequence (35%). Native PAG was purified from Podospora anserina and recombinant PAG was expressed using Pichia pastoris. Both of native and recombinant PAG revealed high specificity for α-1,3- and α-1,4-glucosidic linkages in hydrolysis as well as transglycosylation, while SOG preferred α-1,4-glucosidic linkage in hydrolysis and generated α-1,4- and α-1,6-glucosidic linkages in transglycosylation. In order to elucidate the structural elements responsible for different regioselectivity of two enzymes, mutations were introduced on active site of SOG by site-directed mutagenesis. Several loop structures constructing active site, that is, β→α loop 1 and β→α loop 8 from catalytic (β/α)8 barrel domain and N-loop from N-terminal domain, were identified to be important structural determinants of regioselectivity. A quadruple mutant SOG 231F232/W324Y/Y700E/L701Y showed increase of α-1,3-specificity and decrease of α-1,6-specificity in hydrolysis as well as transglycosylation. Consequently, the present study clarified structure-function relationship of fungal α-glucosidases, especially focused on α-1,6- and α-1,3-glucosidic linkages.