9α-Hydroxyandroster-4-ene-3,17-dione (9-OH-AD) is a representative steroid drug intermediate that can be prepared by phytosterols (PS) biotransformation with mycobacteria in a resting cell-cyclodextrin system. In this study, over-expression of 17β-hydroxysteroid dehydrogenase (Hsd4A) was testified to enhance the side-chain degradation of PS and to reduce the incomplete degradation by-products. Meanwhile, the complete degradation product 4-androstene-3,17-dione (AD) was increased due to lack of 3-Ketosteroid 9α-Hydroxylase (KshA1) activities. To increase the production and purity of 9-OH-AD, the metabolic pathway of the side-chain degradation of PS and 9-position hydroxylation was modulated by balancing the over-expression of Hsd4A and KshA1 in mycobacteria and reducing the bioconversion rate via lowering the ratio of PS and cyclodextrin. The production and purity of 9-OH-AD in broth were improved from 22.18 g/L and 77.13% to 28.27 g/L and 87.84%, with a molar yield of 78.32%.