Polymyxin B (PB), as the last-line of defense against multidrug-resistant Gram-negative bacteria, has caused resistance to P. aeruginosa recently. Fortunately, synergistic treatment could preserve the last class of antibiotics and reduce the emergency of drug resistance. Here, we performed a screen of 970 approved drugs synergized with PB against the P. aeruginosa DK2, which is severely resistant to PB, MIC=512μg/mL. Encouragingly, we found fluoroquinolones could synergy with PB and achieved an obvious reduction in MIC of PB below the clinical susceptible breakpoint (2μg/mL). Especially, gemifloxacin achieved the highest synergistic effect with PB, leading to a 4096-fold MIC reduction (reduced from 512μg/mL to 0.125μg/mL). Furthermore, synergistic effect was also observed in the combination of gemifloxacin and colistin. Finally, outer membrane permeabilization assay showed that gemifloxacin could increase the permeability of bacterial cell membranes for P. aeruginosa which partly explained the synergy mechanism. These results indicate that fluoroquinolones represent attractive synergists to address the emerging threat of polymyxin-resistant infections.