Abstract: The widespread use of antibiotics has led to a global problem of bacterial resistance, weakening the therapeutic effect of antibiotics. To search for new targets for antibiotic action, the bactericidal effects of aminoglycoside antibiotics, β-lactam antibiotics, and quinolone antibiotics on the cysK gene knockout strain（ΔcysK）of Escherichia coli were tested, and the bactericidal effect of gentamicin on the cysK gene overexpressing strain ΔcysK-pCA24N（cysK）of Escherichia coli was further tested. Finally, the bactericidal effect of gentamicin on the cysK gene knockout strain（ΔcysK）of Escherichia coli was tested by exogenous addition of a low concentration of cysteine. The results showed that compared with the wild-type Escherichia coli（WT), the cysK gene knockout strain（ΔcysK）of Escherichia coli had a tolerance phenotype to aminoglycoside antibiotics, β-lactam antibiotics, and quinolone antibiotics, and the cysK gene overexpressing strain ΔcysK-pCA24N（cysK）of Escherichia coli restored its sensitivity to gentamicin. This indicates that the cysK gene plays a role in bacterial resistance to aminoglycoside antibiotics. Exogenous addition of a low concentration of cysteine can restore the sensitivity of the cysK gene knockout strain（ΔcysK）of Escherichia coli to gentamicin, but exogenous addition of high concentration of cysteine will exacerbate the tolerance phenotype of the cysK gene knockout strain（ΔcysK）of Escherichia coli to gentamicin. Therefore, the reason why the knockout of the cysK gene leads to bacterial tolerance to gentamicin may not be simply the lack of cysteine, and further in-depth research is worthy of attention. The above results confirm that the cysK gene has a significant impact on the tolerance of Escherichia coli to gentamicin and can be used as a new antibiotic target.