Aim To examine the synergistic effect of calycosin combined with polymyxin B against various mcr-1-positive bacterial strains. Methods and Results In this study, we found a potential inhibitor of MCR-1, calycosin, that could significantly restore the antibacterial activity of polymyxin B. The synergistic effect of calycosin combined with polymyxin B against various mcr-1-positive bacterial strains was confirmed by checkerboard minimum inhibitory concentration assays, time-kill curve assays and disk diffusion assays. The fractional inhibitory concentration indexes ranged from 0 center dot 15 +/- 0 center dot 03 to 0 center dot 28 +/- 0 center dot 05, and the zones of inhibition increased from 13 center dot 33 +/- 0 center dot 47 to 17 center dot 67 +/- 0 center dot 47 mm with the combined therapy of calycosin and polymyxin B. In addition, the combined therapy significantly reduced the number of bacteria in the medium. However, at the concentrations required for the synergistic effect with polymyxin B, calycosin alone showed no effect on bacterial growth or MCR-1 production. Calycosin treatment exhibited no cytotoxicity to HeLa cells or A549 cells at calycosin concentrations below 32 mu g ml(-1). Conclusions Therefore, our results suggested that calycosin could be used as a potential MCR-1 inhibitor to restore the bactericidal effect of polymyxin B without affecting bacterial viability or existing cytotoxicity. Significance and Impact of the Study The synergistic effect of calycosin combined with polymyxin B against various mcr-1-positive bacterial strains paves the way for future pharmaceutical applications of calycosin in fighting mcr-1-positive bacterial infections.