A hallmark of tuberculosis is the ability of the causative agent, Mycobacterium tuberculosis, to persist for decades despite a vigorous host immune response. Previously, we identified a mycobacterial gene cluster, mce4, that was specifically required for bacterial survival during this prolonged infection. We now show that mce4 encodes a cholesterol import system that enables M. tuberculosis to derive both carbon and energy from this ubiquitous component of host membranes. Cholesterol import is not required for establishing infection in mice or for growth in resting macrophages. However, this function is essential for persistence in the lungs of chronically infected animals and for growth within the IFN-γ-activated macrophages that predominate at this stage of infection. This finding indicates that a major effect of IFN-γ stimulation may be to sequester potential pathogens in a compartment devoid of more commonly used nutrients. The unusual capacity to catabolize sterols allows M. tuberculosis to circumvent this defense and thereby sustain a persistent infection.