The twin-arginine translocation (Tat) system is involved in a variety of important bacterial physiological processes. Conserved among bacteria and crucial for virulence, the Tat system is deemed as a promising anti-microbial drug target. However, the mechanism of how the Tat system functions in bacterial pathogenesis has not been fully understood. In this study, we showed that the Tat system was critical for the virulence of an extra-intestinal pathogenic E. coli (ExPEC) strain PCN033. A total of 20 Tat-related mutant strains were constructed, and competitive infection assays were performed to evaluate the relative virulence of these mutants. The results demonstrated that several Tat substrate mutants, including the Δ sufI , Δ amiA Δ amiC double mutant as well as each single mutant, Δ yahJ , Δ cueO , and Δ napG , were significantly outcompeted by the WT strain, among which the Δ sufI and Δ amiA Δ amiC strains showed the lowest competitive index (CI) value. Results of individual mouse infection assay, in vitro cell adhesion assay, whole blood bactericidal assay, and serum bactericidal assay further confirmed the virulence attenuation phenotype of the Δ sufI and Δ amiA Δ amiC strains. Moreover, the two mutants displayed chained morphology in the log phase resembling the Δ tat and were defective in stress response. Our results suggest that the Tat system and its dependent cell division proteins SufI, AmiA, and AmiC play critical roles during ExPEC pathogenesis.