Background: Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), manipulates the host immune responses for its survival. Several secretary proteins exported through ESX secretion system modulate the antibacterial effector functions of macrophages. Here we attempted to functionally elucidate the role of one of the previously uncharacterized ESAT-6 family protein in Mtb pathogenesis. Methods & Materials: Mtb gene is cloned and expressed in Mycobacterium smegmatis. Invasion assay is performed in HeLa cells where as survival assays were carried out in both mouse and human macrophages. Survival mechanisms were studied by quantifying free radicals formation and host survival rate. Induction of autophagic pathway is studied by the expression of LC3. Nuclear and chromosomal aberrations were studied using micronucleus assay. The level of cytokine expressions were measured using qPCR. Results: Recombinant Mycobacterium smegmatis strain expressing ESAT-6 family protein showed more invasion in human epithelial cells (HeLa) and survival in mouse macrophages (RAW264.7) and human monocyte derived macrophages (THP-1). The survival mechanistic studies revealed, infection induced host cell death and also up regulation of the level of nitric oxide, inducible nitric oxide synthase, reactive oxygen species, superoxide dismutase and catalase activities. Moreover, Mycobacterium smegmatis expressing ESAT-6 infection reduced T-cell survival, caused loss of mitochondrial membrane integrity, activated autophagy and also induced the expression of NF-κB and late endosomal markers such as Rab10, LAMP-2, Cathepsin-B and Cathepsin-Z in macrophages. An up regulation of both pro-inflammatory (TNF-α, IL-12) and anti-inflammatory (IL-10, TGF-β) cytokines was also observed after infection in macrophages. Genome stability studies showed that recombinant Mycobacterium smegmatis strain expressing ESAT-6 family protein infection led to several chromosomal abnormalities such as micronuclei formation in cytokinesis blocked cells, chromosomal malsegregation and nuclear fragmentation. Conclusion: We envision that this study offers a novel mechanism of host cell death by Mtb infection. Taken together, this study has identified a novel ESAT-6 like family protein that acts as virulence as well as an immunomodulatory factor to evade killing by host, and thus it may provide a novel drug target.