In this paper, a novel and unique ginsenoside Rg1-hydrolyzing β-D-Glucosidase from Penicillium sclerotiorum was isolated, characterized, and generally described. The β-Glucosidase is an～180 kDa glycoprotein with pI 6.5, and consists of four identical subunits of ～40 kDa. The β-Glucosidase was active in a narrow pH range (4-5) and at relatively high temperature (60-70℃). The optimal activity against p-nitrophenyl-β-D-glucopyranoside(pNPG) was as follows: pH 4.5 and temperature 65℃. Under these conditions, the Km of the enzyme was 0.715 mM with a Vmax of 0.243 mmol nitrophenol/min mg. Metal ions such as Ba2+, K+, Fe3+, and Co2+ significantly promoted the enzymatic activity, while Ca2+, Mg2+,and Ag+ inhibited its activity. Of the tested substrates,only ginsenoside Rg1 could be specifically hydrolyzed by the β-Glucosidase at the C6-glucoside to form the rare ginsenoside F1. These properties were novel and different from those of other previously described glycosidases.