The global dissemination of antibiotic-resistant Mycobacterium tuberculosis has underscored the urgent need to understand the molecular mechanisms of immunity to this pathogen. Use of biological immunomodulatory compounds to enhance antituberculous therapy has been hampered by the limited efficacy of these agents toward infected human macrophages and lack of information regarding their mechanisms of activity. We tested the hypotheses that extracellular ATP (ATPe) promotes killing of virulent M. tuberculosis within human macrophages, and that activation of a specific macrophage enzyme, phospholipase D (PLD), functions in this response. ATPe treatment of infected monocyte-derived macrophages resulted in 3.5-log reduction in the viability of three different virulent strains of M. tuberculosis. Stimulation of macrophage P2X7 purinergic receptors was necessary, but not sufficient, for maximal killing by primary macrophages or human THP-1 promonocytes differentiated to a macrophage phenotype. Induction of tuberculocidal activity by ATPe was accompanied by marked stimulation of PLD activity, and two mechanistically distinct inhibitors of PLD produced dose-dependent reductions in ATPe-induced killing of intracellular bacilli. Purified PLD restored control levels of mycobacterial killing to inhibitor-treated cells, and potentiated ATPe-dependent tuberculocidal activity in control macrophages. These results demonstrate that ATPe promotes killing of virulent M. tuberculosis within infected human macrophages and strongly suggest that activation of PLD plays a key role in this process.