AbstractIn this study, the ability of CYP109E1 from Bacillus megaterium DSM319 to metabolize cholesterol was investigated. This steroid was identified as a new substrate to be converted by CYP109E1 with adrenodoxin and adrenodoxin reductase as redox partners in vitro. The biotransformation was successfully reproduced in vivo by using Bacillus megaterium cells that overexpressed CYP109E1. To enhance the production of cholesterol derivatives, an Escherichia coli based whole‐cell system that harbored CYP109E1 was established. This novel system showed a 3.3‐fold higher activity than that of the B. megaterium system, yielding about 45 mg L−1 of these products. Finally, the reaction products were isolated and identified to be the highly important cholesterol derivatives 24(S)‐ and 25‐hydroxycholesterol.