Abstract: In order to solve the problem of bacterial resistance caused by the abuse of antibiotics and find new targets for the action of antibiotics, in this study, the bactericidal phenotype differences of wild-type strain BW25113 (WT) and lysR gene knockout strain ( ΔlysR) against aminoglycoside antibiotics (gentamicin, tobramycin, streptomycin), quinolones and β-lactam antibiotics were detected. Furthermore, the bactericidal phenotype of gentamicin against the overexpression strains BW25113-pCA24N and BW25113-pCA24N (lysR) was further tested. Finally, the bactericidal phenotype of gentamicin on the wild-type strain BW25113 (WT) and lysR gene knockout strain (ΔlysR) was detected by the exogenous addition of lysine, so as to determine the importance of lysR gene in bacterial resistance. The results showed that: compared with the wild-type strain BW25113 (WT), the lysR gene knockout strain (ΔlysR) was resistant to aminoglycoside antibiotics, but not to quinolones and β-lactam antibiotics, and the overexpression strain BW25113-pca24N (lysR) could restore the sensitivity to gentamicin. The exogenous addition of lysine could alleviate the gentamicin resistance phenotype of the lysR gene knockout strain (ΔlysR). The above results confirmded that the lysR gene had a significant effect on aminoglycosides resistance in E. coli, and there may be a unique resistance mechanism of lysR gene, which could be used as a new target of antibiotics.