Mycobacterium tuberculosis(Mtb) WhiB3 is an iron–sulfur cluster-containing transcription factor belonging to a subclass of the WhiB-Like (Wbl) family that is widely distributed in the phylum Actinobacteria. WhiB3 plays a crucial role in the survival and pathogenesis ofMtb. It binds to the conserved region 4 of the principal sigma factor (σA4) in the RNA polymerase holoenzyme to regulate gene expression like other known Wbl proteins inMtb. However, the structural basis of how WhiB3 coordinates with σA4to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σA4complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σA4complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNAin vitroand transcriptional regulationin Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression inMtbby partnering with σA4and engaging with DNAviathe subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7.