The ability to form persister cells by Mycobacterium tuberculosis (Mtb) is a prime cause for the emergence of drug-resistant strains. A large number of toxin–antitoxin systems in the Mtb genome are postulated to promote bacterial persistence. The largest family of toxin–antitoxin systems encoded in the genome of Mtb is VapBC, with 47 VapBC toxin–antitoxin systems regulated by VapB antitoxins. In this study, we characterized the structure of VapB46 antitoxin and determined its interaction with its cognate DNA sequence. Using electrophoretic mobility shift assay and DNase I footprinting we showed that VapB46 binds to two sites in the upstream promoter–operator region. Using nuclear magnetic resonance (NMR)-based structural studies we found that VapB46 has a well-folded dimeric N-terminal domain, which contains a Phd/YefM motif and is involved in DNA binding. The remaining C-terminal residues are disordered but promote higher order oligomerization of VapB46. We propose a DNA-binding model in which tetrameric VapB46 binds to the two sites in its promoter–operator region, with each site bound by its dimeric N-terminal domain.