Abstract The transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown BrlR plays an important role in high level drug tolerance of P. aeruginosa in biofilm. Its drug tolerance ability can be enhanced by 3′,5′-cyclic diguanylic acid (c-di-GMP). Here, we show the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.