Plasmid-mediated transmission of the gene significantly impairs the antimicrobial activity of colistin, limiting clinical therapeutic options. In this study, we evaluated the potential of bosutinib in restoring the susceptibility of colistin to mcr-1-positive drug-resistant using a "drug repurposing" strategy and explored its mechanism of action to develop a new combination therapy regimen. We found that bosutinib combined with colistin significantly restored the susceptibility of mcr-1-positive to colistin and showed strong bactericidal and antibiofilm activities, confirmed by drug sensitivity assays, viable bacterial counts, and biofilm assays. Meanwhile, membrane permeability assay, reactive oxygen species (ROS) measurement, molecular docking, and SPR analysis showed that bosutinib could enhance bacterial membrane permeability, increase ROS accumulation, and directly bind to the MCR-1 protein, disrupting its resistance mechanism. Furthermore, in an infected animal model, bosutinib combined with colistin significantly increased the survival and reduced the bacterial load in tissues, confirming its in vivo antimicrobial efficacy. In conclusion, the present study reveals that bosutinib restores the antimicrobial activity of colistin through dual mechanisms: membrane permeability enhancement and direct targeting of the MCR-1 protein. Indeed, the discovery of bosutinib not only expands the application of tyrosine kinase inhibitor analogues in the field of anti-infective drugs but also provides a potentially new alternative for the clinical treatment of MCR-1-positive bacterial infections.