Abstract: Androsten-1, 4-diene-3, 17-dione (ADD) was an irreplaceable intermediate and a raw material for steroid hormone drugs. A strain of ADD producing bacteria was obtained by overexpressing 3-sterosterone-Δ¹-dehydrogenase (KstD) in Mycobacterium sp. ZFZ. Subsequently, the effects of different media compositions on the production of ADD by this strain were studied. On the basis of single factor experiments, ADD production as index response surface analysis was applied to optimize the fermentation medium composition. The quadratic regression analysis was applied to get the optimal level of main factors. The results showed that the most suitable transformation culture conditions were corn steep liquor 23 g/L, glucose 9 g/L, NaNO₃ 2.8 g/L and K₂HPO₄ 2 g/L. The yield of ADD reached (4.12±0.18) g/L when the concentration of substrate sterol was 10 g/L in the optimal condition. And the yield of ADD increased nearly 18 times than Mycobacterium sp. ZFZ.