In this study, a method for the efficient production of dehydroepiandrosterone (DHEA) from phytosterols in a vegetable oil/aqueous two-phase system by Mycobacterium sp. was developed. After the 3-hydroxyl group of phytosterols was protected, they could be converted into DHEA with high yield and productivity by Mycobacterium sp. NRRL B-3683. In a shake flask biotransformation, 15.05gl(-1) of DHEA and a DHEA yield of 85.39% (molmol(-1)) were attained after 7days with an initial substrate concentration of 25gl(-1). When biotransformation was carried out in a 30-l stirred bioreactor with 25gl(-1) substrate, the DHEA concentration and yield was 16.33gl(-1) and 92.65% (molmol(-1)) after 7days, respectively. The results of this study suggest that inexpensive phytosterols could be utilized for the efficient production of DHEA.