ABSTRACT The ParE family of toxins is known to target Gyrase through an as yet unknown molecular mechanism. Here we show that Vibrio cholerae ParE2 ( Vc ParE2) interacts with Gyrase in a DNA-dependent manner. The interaction site is located at the ParE-specific α-hairpin and involves Trp25 as a key residue. The latter is poorly conserved within the ParE family, although full toxicity is only retained upon substitution with Tyr in the Vc ParE2 context. In vitro , the Trp25Ala mutation reduces the rate of Gyrase-mediated supercoiling, while the so-called cleavable complex remains stabilized. The C-terminal domain of the antitoxin Vc ParD2, which wraps its intrinsically disordered domain around Vc ParE2 without covering the Trp25-containing interaction site, binds to Vc ParE2 with high affinity, but only partially prevents ParE-mediated growth inhibition. Full inhibition of Vc ParE2 requires full length Vc ParD2 that leads to a complex where Trp25 is fully shielded from solvent.