Universally conserved signal recognition particle (SRP) pathway that mediates co-translational targeting of membrane and secretory proteins is essential for eukaryotic and prokaryotic cells. The Mycobacterium tuberculosis SRP pathway consists of two proteins Ffh, FtsY and a 4.5S RNA molecule. Although the Escherichia coli SRP pathway is well studied, understanding about the M. tuberculosis SRP pathway components is very limited. In this study, we have over-expressed and characterized the M. tuberculosis SRP receptor (SR) FtsY as a GTP binding protein. Further, we established the direct protein-protein interaction between Ffh and FtsY. The Ffh-FtsY complex formation resulted in mutual stimulation of their GTP hydrolysis activity. We also attempted to biochemically characterize the SRP components by constructing the antisense gene knockdown strains of ffh and ftsY in M. tuberculosis. Loss of ffh and ftsY resulted in decreased in vitro growth rate of antisense ffh strain as compared to antisense ftsY strain. Finally, the 2D-gel electrophoresis of antisense depleted ffh and ftsY strains identified differential expression of 14 proteins.