The secreted Mycobacterium tuberculosis protein, ESAT6, has been studied extensively in pathogenicity and vaccine experiments. Despite these studies little is known about the function of this protein. In this report, we demonstrate that ESAT6 induces apoptosis in THP-1 human macrophages using fluorescein isothiocyanate-Annexin V and intracellular caspase staining. We show that the induction of apoptosis by ESAT6 is dependent on the dose of the protein and the expression of caspase genes. Using real-time RT-PCR, we found that expression of caspase-1, -3, -5, -7 and -8 genes was upregulated in cells treated with ESAT6 relative to untreated cells. Furthermore, we show that while infection of THP-1 cells with wild-type M. tuberculosis strain H37Rv resulted in significant apoptosis 48 h post infection, a deletion mutant that does not express ESAT6 failed to induce significant apoptosis. Finally, experimental results using a cell impermeable fluorescent stain suggests that the formation of membrane pores may be a primary mechanism by which ESAT6 evokes an apoptotic response.