Effects of different carbon sources on growth, membrane permeability, β-sitosterol consumption, androstadienedione and androstenedione (AD(D)) production by Mycobacterium neoaurum were investigated. The results indicated that glucose was advantageous to the growth and resulted in the adverse effects on the phytosterols consumption and AD(D) production compared to the results of propanol and isopropanol as sole carbon source. The cell wall widths of 9.76 by propanol and 8.00 nm by isopropanol were 38.3% and 49.4% thinner than that of 15.82 nm by glucose, respectively. The partition coefficient of the cell grown in propanol and isopropanol were 18.1 and 22.2, which were 7.23 and 9.09 folds higher than that of the cell grown in glucose.