Cholesterol catabolism is widespread in actinobacteria and is critical forMycobacterium tuberculosis(Mtb) virulence. Catabolism of steroid nucleus rings C and D is poorly understood: it is initiated by the CoA thioesterification of 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) by FadD3, whose gene is part of the KstR2 regulon. InMtb,genes of this regulon were upregulated up to 30- and 22-fold during growth on cholesterol and HIP, respectively, versus another minimal medium. In contrast, genes involved in degrading the cholesterol side-chain and nucleus rings A and B were only upregulated during growth on cholesterol. Similar results were obtained inRhodococcus jostiiRHA1. Moreover, the regulon was not upregulated in a ΔfadD3mutant unable to produce HIP-CoA. In electrophoretic mobility shift assays, HIP-CoA relieved the binding of KstR2Mtbto each of three KstR2 boxes: CoASH, HIP and a related CoA thioester did not. Inspection of the structure of KstR2RHA1 revealed no obvious HIP-CoA binding pocket. The results establish thatMtbcan catabolize the entire cholesterol molecule and that HIP-CoA is an effector of KstR2. They further indicate that KstR2 specifically represses the expression of the HIP degradation genes in actinobacteria, which encode a lower pathway involved in the catabolism of multiple steroids.