Mitochondria are the powerhouse of the cell and a critical cell signalling hub that decides the fate of the cell. Mycobacterium tuberculosis (Mtb) being a successful pathogen targets and controls the host mitochondria for pathogenesis. Various effector proteins of Mtb are also known to target host mitochondria which include few proteins of a unique Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) family exclusively present in pathogenic mycobacteria, but many of them are still uncharacterized. The present study investigates one such late expressing Rv0109 (PE_PGRS1) protein of Mtb. In-silico analysis predicted the presence of mitochondria targeting signal sequences in Rv0109 and its role in regulation of cysteine type endopeptidase (caspase) activity during apoptosis. Recombinant Rv0109 gets localized to mitochondria of THP1 macrophages as shown by confocal microscopy. Rv0109 was observed to induce mitochondrial stress which resulted in mitochondrial membrane depolarization, upregulation of mitochondrial superoxides and release of Cytochrome-C in the cytoplasm through flow cytometry. Depleted intracellular ATP was observed in THP1 macrophages in response to Rv0109. This mitochondrial stress in response to Rv0109 was observed to culminate in increased expression of pro-apoptotic Bax and Bim factors and caspase activation leading to macrophage apoptosis. Since Rv0109 is a late stage specific protein expressed within granuloma; mitochondria mediated apoptosis induced by Rv0109 may be explored for its role in granuloma maintenance and pathogen persistence.