The human pathogen Mycobacterium tuberculosis requires the ESX-1 secretion system for full virulence. EspR plays a key role in ESX-1 regulation via direct binding and transcriptional activation of the espACD operon. Here, we describe the crystal structures of EspR, a C-terminally truncated form, EspR Delta 10, as well as an EspR-DNA complex. EspR forms a dimer with each monomer containing an N-terminal helix-turn-helix DNA binding motif and an atypical C-terminal dimerization domain. Structural studies combined with footprinting experiments, atomic force microscopy and molecular dynamic simulations allow us to propose a model in which a dimer of EspR dimers is the minimal functional unit with two subunits binding two consecutive major grooves. The other two DNA binding domains are thus free to form higher-order oligomers and to bridge distant DNA sites in a cooperative way. These features are reminiscent of nucleoid-associated proteins and suggest a more general regulatory role for EspR than was previously suspected.