The 9 alpha-hydroxyandrost-4-ene-3,17-dione (9 alpha - OH-AD) is a key intermediate for synthetizing steroid pharmaceutical compounds. It is bioconverted by Mycobacterium sp. LY-1 from phytosterols. During biotransformation, the low solubility of phytosterols in water limits 9 alpha - OH-AD production. In the present work, polyoxyethylene (10) nonylphenyl ether (TX-40), a nonionic surfactant, was first used to enhance phytosterol solubility during 9 alpha - OH-AD biotransformation. In the presence of 0.7% TX-40, phytosterol solubility was increased 12.1-fold, and the 9 alpha - OH-AD molar yield reached 42.5% at the phytosterols concentration of 15 g/L, an increase of 217.2% over controls. The improvement is sufficient to replace the traditional process of adding soybean oil. Scanning electron microscopy showed that the extracellular membrane-like structure was loosened by TX-40, but cells maintained their integrity. The fatty acid profile of Mycobacterium sp. LY-1 cells revealed that TX-40 altered the composition of the cell membrane and improved its permeability. The transcript levels of key enzymes involved in cell membrane lipid synthesis were downregulated significantly upon TX-40 addition compared with controls. These results demonstrated that the increase in product molar yield by TX-40 was primarily related to easier substrate transportation across the cell membrane of Mycobacterium sp. LY-1.