To investigate the effect and the mechanism of gene deletion on the drug susceptibility of uropathogenic producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People’s Hospital, meanwhile its gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (=-2.121,<0.05;=-2.236,<0.05;=-2.236,<0.05;=-2.121,<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (=0,0.05). The expression levels of ESBLs-enconding genes and in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene in Δpk1 was significantly up-regulated, while the expression of and were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes , and were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.