Objective To investigate the mechanisms through which gentamicin at a subinhibitory concen⁃tration inhibits the swarming of E. coli. Methods To confirm the inhibitory effect of pretreated gentamicin on theswarming of E. coli ATCC25922，real⁃time PCR was used to determine the effect of gentamicin on the expressionof tricarboxylic acid cycle（TCA）related genes，and ATP detection kit was used to detect the effect of gentamicinon cellular ATP production. The effect of ATP on the swarming of gentamicin⁃pretreated E. coli was observed.Results The pretreated gentamicin with 1/2MIC significantly inhibited the swarming of Escherichia coli ATCC25922.By Real⁃time PCR，TCA⁃related genes of E. coli treated with gentamicin were significantly down⁃regulated. Theexpressions of poxB，aceE，gltA，sdhD，sdhC，sucC，fumA and icd genes were down⁃regulated，and the productionof cellular ATP of E. coli ATCC25922 decreased after pretreatment. The inhibition of gentamicin on the swarming ofE. coli could be reversed by adding ATP in the swarming plate. Conclusion Gentamicin inhibits the swarming ofE. coli by downregulating TCA related genes and reducing the production of cellular ATP. Our findings provide anew theoretical basis for revealing the mechanisms of the post⁃antibiotic effect of gentamicin.