Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin–antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.