The recent emergence of the plasmid-mediated colistin resistance gene mcr-1 poses a substantial clinical threat to the severe infections caused by CRE (Carbapenem Resistant Enterobacteriaceae), as the treatment failure of the mcr-1-positive CRE “Superbug” most likely occurs by using the combination of carbapenem and polymixins. Therefore, our study aims to seek a potent MCR-1 inhibitor to fight this infection. A checkerboard MIC (Minimum Inhibitory Concentration) assay, time-killing assay, MPNP (Modified rapid polymyxin Nordmann/Poirel) test, combined disk test and molecular modelling analysis were performed on different mcr-1-positive strains to confirm the synergistic effects of the combination of colistin and osthole (OST). And a thigh mouse infection model was also used to evaluate such synergies. We identified that OST regained the bactericidal activity of polymyxins (FIC (Fractional Inhibitory Concentration) index = 0.11±0.04 − 0.29±0.10) against mcr-1-positive Enterobacteriaceae including Escherichia coli and Klebsiella pneumoniae. The in-vitro time-killing assays showed that either OST or polymyxins failed to eradicate mcr-1-positive Enterobacteriaceae, but the combination eliminated mcr-1-positive Enterobacteriaceae by 3–7-h post-inoculation. The mouse infection model demonstrated that the combination therapy significantly reduced the bacterial load in the thighs following subcutaneous administration. Our results established that OST is a promising natural compound that could be used to extend the life of polymyxins and to tackle the inevitability of serious infections caused by polymyxin-resistant bacteria.