To enhance the yield of 9 alpha-hydroxy-4-androstene-3,17-dione (9-OHAD) from phytosterols, a phytosterol transport system was constructed in Mycobacterium sp. strain MS136. 9-OHAD can be produced via the controlled degradation of phytosterols by mycobacteria. This involves an active transport process that requires trans-membrane proteins and ATP. A phytosterol transport system from Mycobacterium tuberculosis H37Rv was constructed in Mycobacterium sp. strain MS136 by co-expression of an energy-related gene, mceG, and two integrated membrane protein genes, yrbE4A and yrbE4B. The resultant of the Mycobacterium sp. strain MS136-GAB gave 5.7 g 9-OHAD l(-1), which was a 20% increase over 4.7 g l(-1) by the wild-type strain. The yield of 9-OHAD was increased to 6.0 g l(-1) by optimization of fermentation conditions, when 13 g phytosterols l(-1) were fermented for 84 h in 30 ml biotransformation medium in shake flasks. Phytosterol transport system plays an active role in the uptake and transport of sterols, cloning of the system improved the mass transfer of phytosterols and increased the production of 9-OHAD.