The β-D-glucosidase (EC 3. 2. 1. 21), a member of cellulolytic enzyme family, was isolated from the culture filtrate of Aspergillus phoenicis, OM 329, and was purified partially by DEAE-cellulose ion-exchange chromatography and by an affinity column employing p-nitrophenyl-1-thin-β-D-glucoside as a ligand on Sepharose 4B matrix. The enzyme showed a very limitted substrate specificity toward non-reducing terminal 1-β-D-glucopyranosyl linkage. The Km values for cellobiose and p-nitrophenyl-β-D-glucoside (PNPG) were obtained as 3.5×10^(-4) M and 7.5×10^(-4) M, respectively. β-D-Glucose showed a product inhibition, but no inhibition was found with p-nitrophenol. The enzyme also showed its transglucosvlase activity, and this suggest a possible involvement of a glucosyl-enzyme intermediate during the enzyme catalysis. Marked inhibition by nojirimycin and hydroxylamine may also suggest a pseudo-covalent glucosyl-enzyme intermediate. An information on the functional groups at the enzyme active center was obtained from a pH-rate profile and chemical modification experiments, and a model mechanism of the enzyme reaction was suggested based on this and other kinetic analysis.