Persistent Mycobacterium tuberculosis (MTB) likely encounters a phosphate-limited environment within macrophage phagosomes. We studied MTB growth, antibiotic susceptibility, and gene expression during phosphate limitation. With use of MTB mutants deficient in phosphate-related genes, we assessed bacillary survival under phosphate-limited conditions and in mouse and guinea pig lungs. Phosphate limitation restricted MTB growth in a dose-dependent manner, and phosphate-starved bacilli became phenotypically tolerant to isoniazid. The MTB genes ppk1 and relA were upregulated significantly after phosphate starvation, consistent with inorganic polyphosphate accumulation and MTB stringent response induction. The phosphate-specific transport operon pstS3-pstC2-pstA1 was induced during phosphate starvation and its expression was dependent on the 2-component regulatory system SenX3-RegX3. The MTB gene regX3 appears to be essential for bacillary survival during phosphate limitation and in mammalian lungs. Our data suggest that MTB encounters phosphate-limited conditions during mammalian lung infection and that expression of the phosphate starvation response (PSR) is important for MTB persistence.