Pyrazinamide (PZA) is a critical component of first- and second-line treatments of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate Mycobacterium bovis BCG mutants resistant to pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA resistant strains. Rather than finding mutations in the proposed candidate targets fatty acid synthase I and ribosomal protein S1, we found resistance conferring mutations in two pathways: missense mutations in aspartate decarboxylase panD, involved in the synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations in the vitro nonessential polyketide synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM). Probing for cross resistance to two structural analogs of POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM- but not the CoA-related mechanism of action with POA. We demonstrated that POA depletes CoA in wild-type bacteria, which is prevented by mutations in panD. Sequencing 10 POA-resistant Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least 2 distinct mechanisms of resistance to the drug. The emergence of resistance through the loss of a virulence factor in vitro may explain the lack of clear molecular patterns in PZA-resistant clinical isolates, other than mutations in the prodrug-converting enzyme. The apparent interference of POA with virulence pathways may contribute to the drug’s excellent in vivo efficacy compared to its modest in vitro potency.