Through a combination of twice DEAE chromatography by NaCl stepwise and gradient elution with gel filtration chromatography, a kind of ginsenoside-Rb1 hydrolase from crude Helix snailase was separated. The hydrolase was purified to apparent homogeneity on SDS-PAGE. It was estimated that the purified hydrolase was consisted of four identical subunits with a molecular mass of 110-115 kD by SDS-PAGE and gel filtration chromatography. The Km and Vmax values for ginsenoside-Rb1 were calculated to be 0.790 mmol/L and 10.192 micromol/(min x mg) of protein respectively. The ginsenoside-Rb1 hydrolase could only hydrolyze the glycosidic bond at the C20 position of ginsenoside-Rb1 into ginsenoside-Rd.