In this paper, a novel and unique ginsenoside Rg(1)-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°C). The optimal activity against p-nitrophenyl-β-D-glucopyranoside (pNPG) was as follows: pH 4.5 and temperature 65°C. Under these conditions, the K(m) of the enzyme was 0.715 mM with a V(max) of 0.243 mmol nitrophenol/min mg. Metal ions such as Ba(2+), K(+), Fe(3+), and Co(2+) significantly promoted the enzymatic activity, while Ca(2+), Mg(2+), and Ag(+) inhibited its activity. Of the tested substrates, only ginsenoside Rg(1) could be specifically hydrolyzed by the β-glucosidase at the C6-glucoside to form the rare ginsenoside F(1). These properties were novel and different from those of other previously described glycosidases.