The irrational use of broad-spectrum antibiotics has led to increasing resistance of bacteria to antibiotics, and the emergence of the plasmid-mediated colistin resistance gene mcr-1 has led to the dilemma of infections with no available cure. Here, we have found a potential MCR-1 inhibitor for use against infections caused by MCR-1 positive resistant bacteria. A checkerboard MIC (minimum inhibitory concentration) assay, growth curve assay, kill curve assay, cytotoxicity assay, molecular dynamics simulation analysis, Western blot assay and mouse pneumonia model in vivo protection rate assay were used to evaluate the synergy effect between genistein and polymyxins. The results showed that genistein could restore the bactericidal activity against MCR-1-positive strains for which there was no antibacterial activity, and reduce the bacterial load to some extent. Genistein does not inhibit the expression of MCR-1, but inhibits the binding of MCR-1 to its substrate by binding to the amino acids of the active region of MCR-1, thereby inhibiting the biological activity of MCR-1. The in vivo results also showed that the protection rate of mice treated with the combination therapy of genistein and polymyxins increased by 20% compared to that of mice treated with polymyxins alone. Our results confirm that genistein is an inhibitor of MCR-1 and promote its potential use in combination with polymyxins to treat severe infections caused by MCR-1 positive Enterobacteriaceae.