The b-D-glucosidase involved in the biotransformation of swertiamarin to 5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano[3, 4-c] pyridine-1-one and erythrocentaurin was purified and characterized. The enzyme had a molecular weight of 88 kDa as determined by SDS polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 55-60°C and pH 5.0. The enzyme was stable at pH 5-7 and 60°C, and specified on b–(1-4)-glucosidic linkage. The affinity constants for r-NPG and swertiamarin were 0.58 and 0.26 (mM),respectively. So the catalytic constants for r-NPG and swertiamarin were 186.8 and 79.6 (µmol min^-1 mg^-1), respectively. Ca²⁺, Cu ²⁺, Zn²⁺, and Fe³⁺ (0~120 mM) inhibited the b-D-glucosidase, while Mg²⁺ and Mn²⁺ (each 0~120 mM) activated this enzyme. Glucose had a high inhibitory effect on the enzyme and the inhibition constant was 0.13 and thus the concentration of glucose in the broth for producing the b-D-glucosidase should be strictly controlled no more than 8mg/l. The metabolic pathway and the role of the purified b-D-glucosidase were fully discussed.