Abstract: As an important steroid intermediate, 22-hydroxy-23, 24-bisnorchola-1, 4-diene-3-ketone (HPD) was the raw material for the synthesis of many Sebaceous drugs.We overexpressed 3-ketosteroid-Δ¹-dehydrogenase (KstD) gene in Mycobacterium neoaurum DSM 1381, the purity of HPD in fermentation product was increased from 71% to 84%. On the basis of single factor selection, the yield of product HPD was taken as the measurement index, response surface method was used to optimize the fermentation medium, the quadratic regression equation for the change of each influencing factor was established. The final results showed that the most suitable fermentation medium composition conditions were corn steep liquor 9 g/L, NaNO₃ 1.8 g/L, glucose 6 g/L, and K₂HPO₄ 2 g/L.Under this condition, the yield of HPD could reach 3.73 g/L when the concentration of phytosterol was 5 g/L, and the yield of HPD was nearly 2.7 times higher than Mycobacterium neoaurum DSM 1381, it had potential industrial application value.