Aims The aim of this work was to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3 by three thermostable glycosidases. Methods and Results The GH1 thermostable beta-glucosidase Tpebgl1 from Thermotoga petrophlia was found to have the ability to convert ginsenosides Rb1 and Rb2. Its properties concerning ginsenoside conversion were systematically investigated. It had high specific activity on pNPG (162 center dot 20 U mg(-1)) and pNPArp (22 center dot 14 U mg(-1)). The Km and Vmax of Tpebgl1 for pNPG were 0 center dot 28 mmol l(-1) and 470 center dot 2 U mg(-1) and for pNPArp were 17 center dot 30 mmol l(-1) and 74 center dot 28 U mg(-1). Therefore, it could successfully convert ginsenosides Rb1 and Rb2 into ginsenoside Rd, which has been proven by experiments in this paper then. Tpebgl1 also had good tolerance to glucose and some organic solvents. These made Tpebgl1 a good catalyst candidate for industrial application. Finally, it was applied to convert ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, combined with thermostable ginsenoside Rc converting alpha-1,6-l-arabinofranosidase Tt-Afs and ginsenoside Rd converting beta-glucosidase Tpebgl3. A quantity of 10 g l(-1) of ginsenoside extract was transformed into 3 center dot 93 g l(-1) of Rg3 at 90 degrees C, pH 5 center dot 0 for 3 h, with a corresponding molar conversion of 98 center dot 19%. Conclusion The thermostable enzyme Tpebgl1 was found to be a ginsenoside-converting enzyme and successfully applied in the preparation of ginsenoside 20(S)-Rg3 from ginsenoside extract. The three-step cooperate transformation system of ginsenoside extract was established by using Tpebgl1, Tt-Afs (a thermostable ginsenoside Rc converting alpha-1,6-l-arabinofranosidase) and Tpebgl3 (a thermostable ginsenoside Rb1 converting beta-glucosidase). Significance and Impact of the Study Converting all the major ginsenosides into protopanaxadiol-type ginsenoside extract would greatly reduce the cost of ginsenoside Rg3 preparation. Enzymes from thermophilic bacteria can meet the requirement of higher reaction temperatures in industrial reactions for substrate solubility promotion and bacterial contamination prevention.