Summary Degradation of the cholesterol side‐chain in M ycobacterium tuberculosis is initiated by two cytochromes P 450, CYP125A1 and CYP142A1 , that sequentially oxidize C 26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from M ycobacterium smegmatis mc 2 155. Heterologously expressed, purified CYP 125 A 3 and CYP142A2 bound cholesterol, 4‐cholesten‐3‐one, and antifungal azole drugs. CYP 125 A 3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4‐cholesten‐3‐one to the C ‐26 alcohol and subsequently to the acid. The X ‐ray structures of both substrate‐free CYP125A3 and CYP 142 A 2 and of cholest‐4‐en‐3‐one‐bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M . tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3 . In contrast to M . tuberculosis , the M . smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.