Given the important role of polymyxin B (PB) in the treatment of drug-resistant Gram-negative bacterial infections, the emergence of PB resistance poses a serious threat to public health. Adjuvant development is a supplementary strategy that can compensate for the lack of novel antibiotics by protecting PB. In this study, we found a small molecule named Lyb24 that showed weak antibacterial activity (minimum inhibitory concentration ≥ 10 μg/ml) but potentiated and revitalized the efficacy of PB against Gram-negative pathogens, including mcr-1- and mgrB-deletion-mediated PB-resistant strains. Our results showed that Lyb24 inhibits the translational levels of genes associated with the modification of lipid A. In addition, Lyb24 increases the permeability, disrupts the integrity and induces the depolarization of the membrane. We further found that both Lyb24 and PB could directly bind to AzoR and inhibit its activity. Structural analysis showed that Lyb24 binds to the isoalloxazine ring of flavin mononucleotide (FMN) through pi-pi stacking and loop η4 of AzoR. A pneumonia model was used to confirm that the activity against clinical PB-resistant Klebsiella pneumoniae was enhanced due to Lyb24 on PB. In conclusion, we provide a potential therapeutic regimen by combining Lyb24 and PB to treat Gram-negative-resistant bacterial infections. Our findings not only explain the synergistic effect of Lyb24, but also expand our knowledge on the mechanism of action of PB.