Abstract: With the widespread dissemination of the plasmid-mediated colistin resistance gene mcr-1, colistin resistance has spread globally, severely threatening the last line of defense in global anti-infective therapy. This study aimed to evaluate the resistance-reversing effects of linalool combined with colistin against mcr-1-positive Escherichia coli (E. coli) and to elucidate the underlying mechanisms, aiming to propose a therapeutic strategy to combat colistin resistance. The synergistic antibacterial effects in vitro were determined using the broth microdilution checkerboard method and time-kill curves, and biofilm assays. Further investigations elucidated the underlying mechanisms by assessing inner/outer membrane permeability, membrane potential (ΔΨ), dehydrogenase activity (DHA), proton gradient (ΔpH), ATP levels, and mcr-1 gene expression. Finally, we assessed the in vivo efficacy of linalool in combination with colistin against Galleria mellonella infection and mouse acute peritonitis models. Results demonstrated that linalool-colistin combination exhibited synergistic antibacterial effects against mcr-1-positive E. coli both in vitro and in vivo. Mechanistically, linalool directly damaged the bacterial outer membrane, reduced ΔΨ, and increased inner membrane permeability. Moreover, linalool inhibited DHA activity, disrupted ΔpH establishment, ultimately impairing ATP production. Additionally, linalool significantly downregulated the mcr-1 expression, suppressing lipid A modification in resistant strains. These effects collectively enhanced colistin penetration, triggering membrane disruption and energy metabolism collapse, ultimately achieving synergistic antibacterial efficacy. In vivo, combination therapy significantly improved survival rates in both infection models. In conclusion, linalool restores colistin susceptibility in mcr-1-positive E. coli through synergistic mechanisms, providing a promising therapeutic strategy against colistin resistance. All animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Committee of Xiangnan University (approval No.: 2023DWLL029).