Steroids and their oxyfunctionalized counterparts are valuable compounds for the pharmaceutical industry; however, the regio- and stereoselective introduction of oxygen is a challenging task for the synthetic chemistry. Thus, cytochromes P450 play an important role for the functionalization of steroidal compounds. In this study, we elucidated the main product of 11-deoxycorticosterone conversion formed by CYP260B1 from Sorangium cellulosum So ce56 as 9α-OH 11-deoxycorticosterone by NMR spectroscopy. This is, to the best of our knowledge, the first identification of a 9α-hydroxylase for this substrate. In addition, the major side product was identified as 21-OH pregna-1,4-diene-3,20-dione. Studies using 1α-OH 11-deoxycorticosterone as substrate suggested that the major side product is formed via dehydrogenation reaction. This side reaction was considerably decreased by employing the CYP260B1-T224A mutant, which showed an increased selectivity of about 75% compared to the 60% of the wild type for the 9α-hydroxylation. To scale up the production, an E. coli based whole-cell system harboring the CYP260B1-T224A variant as well as two heterologous redox partners was used. Employing growing cells in minimal medium led to a productivity of about 0.25 g/l/d at a 50 ml scale showing the biotechnological potential of this system.