New approaches are required to combat Mycobacterium tuberculosis (Mtb), especially the multi-drug resistant and extremely drug resistant organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3β-hydroxysterols to 3-ketosteroids, but the enzymes responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3β-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD⁺ as a cofactor and oxidizes cholesterol, pregnenolone, and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD⁺ binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (k_cat/K_m) of 3.2 ± 0.4 and 3.9 ± 0.9 μM^-1 min^-1, respectively, at pH 8.5, 150 mM NaCl, 30 mM MgCl₂, and saturating NAD⁺. Trilostane is a competitive inhibitor of dehydroepiandrosterone with a K_i of 197 ± 8 μM. The expression of the 3β-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3β-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with the Rv1106c gene being the one responsible for 3β-hydroxysterol oxidation in Mtb.