Mycobacterium tuberculosis H37Rv contains the kshA (Rv3526) and kshB (Rv3571) genes, encoding 3-ketosteroid 9α-hydroxylase (KSH). Consistent with their predicted roles, the ΔkshA and ΔkshB deletion mutants of M. tuberculosis H37Rv were unable to use cholesterol and 4-androstene-3,17-dione as primary carbon and energy sources. Interestingly, ΔkshA and ΔkshB mutants were also unable to metabolize the steroid substrate 5α-androstane-3,17-dione, whereas wild-type M. tuberculosis H37Rv could. The deletion of either of these genes lead to rapid death of the microorganism in murine infection models and in macrophages, showing that kshA and kshB are essential factors for M. tuberculosis pathogenesis. Penta-acylated trehalose (PAT) biosynthesis was altered in the ΔkshB mutant, but not the ΔkshA mutant. The ΔkshB mutant synthesizes all other types of lipids. The ΔkshB mutant had a thickened outer layer in its cell wall. KshB thus appears to be involved in multiple processes, probably as a reductase of different oxygenases. We conclude that an impaired 3-ketosteroid 9α-hydroxylase activity is the cause of the highly attenuated phenotype of our M. tuberculosis H37Rv mutants.