A novel dioscin-glycosidase that specifically hydrolyzes multi-glycosides, such as 3-O-${alpha}$-L-($1{ o}4$)-rhamnoside, 3-O-${alpha}$-L-($1{ o}2$)-rhamnoside, 3-O-${alpha}$-L-($1{ o}4$)-arabinoside, and ${eta}$-D-glucoside, on diosgenin was isolated from the Absidia sp.d38 strain, purified, and characterized. The molecular mass of the new dioscin-glycosidase is about 55 kDa based on SDS-PAGE. The dioscin-glycosidase gradually hydrolyzes either 3-O-${alpha}$-L-($1{ o}4$)-Rha or 3-O-${alpha}$-L-($1{ o}2$)-Rha from dioscin into 3-O-${alpha}$-L-Rha-${eta}$-D-Glc-diosgenin, further rapidly hydrolyzes the other ${alpha}$-L-Rha from 3-O-${alpha}$-L-Rha-${eta}$-D-Glc-diosgenin into the main intermediate products of 3-O-${eta}$-D-Glc-diosgenin, and subsequently hydrolyzes these intermediate products into aglycone as the final product. The enzyme also gradually hydrolyzes 3-O-${alpha}$-L-($1{ o}4$)-arabinoside, 3-O-${alpha}$-L-($1{ o}2$)-rhamnoside, and ${eta}$-D-glucoside from [3-O-${alpha}$-L-($1{ o}4$)-Ara, 3-O-${alpha}$-L-($1{ o}4$)-Rha]-${eta}$-D-Glc-diosgenin into diosgenin as the final product, exhibiting significant differences from previously reported glycosidases. The optimal temperature and pH for the new dioscin-glycosidase is $40^{circ}C$ and 5.0, respectively. Whereas the activity of the new dioscin-glycosidase was not affected by $Na^+$, $K^+$, and $Mg^{2+}$ ions, it was significantly inhibited by $Cu^{2+}$ and $Hg^{2+}$ ions, and slightly affected by $Ca^{2+}$ ions.