[Objective] To study the role of response regulator rstA of TCS RstA/RstB in pathogenesis of uropathogenic Escherichia coli (UPEC). [Methods] By using λ Red recombination system, we generated the rstA knockout mutant U17ΔrstA and the complementation strain ReU17ΔrstA. We then compared and analyzed the characterization of the mutant strain and the wild type strain in vivo and in vitro.[Results] The growth curves in LB showed that the deletion of rstA did not affect growth kinetics of mutant, whereas in LB-Fe medium, the growth rate of U17ΔrstA was lower than that of the wild-type strain U17. Under the selected environmental stress conditions in vitro, the bacterial survival experimental results showed that the mutant was not sensitive to acid, alkali, high osmotic pressure, urea and oxidants. Strain biofilm assay showed that the biofilm formation ability of the mutant was similar to that of the wild-type strain. qRT-PCR results showed that the rstA gene was significantly upregulated in LB-Fe medium, indicating that the iron-deficiency environment may be the stimulus signal evoking the RstA regulator. The mouse model of ascending urinary tract infection demonstrated that the deletion of rstA led to attenuation of virulence, because the mutant showed significantly decreased colonization compared with the wild type strain in urine, bladder and kidney, whereas the complementation strain restored the virulence to resemble that of wild-type strain. [Conclusion] The rstA gene was a potential virulence factor and associated with the pathogenesis of UPEC.