Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.