Phenazine-1-carboxylic acid (PCA), a member of phenazines secreted by microorganisms, inhibits the growth of many bacteria and fungi. Xanthomonas campestris pv. campestris is the causal agent of black rot, the most important disease of cruciferous crops worldwide, and is more tolerant to PCA than other Xanthomonas species. Previous studies reported that reactive oxygen species (ROS) scavenging ability is involved in regulating the PCA tolerance of Xanthomonas species. Additionally, the cytochrome c maturation (CCM) system has been found to play a more important role in tolerance to phenazines than the ROS scavenging system. In this study, a highly PCA-sensitive insertion mutant of X. campestris pv. campestris, X-5, was identified and studied. The insertion site of X-5 was found to be in tatB gene (XC_4183), which encodes a subunit of the twin-arginine translocation (TAT) complex. Disruption of the three genes of TAT pathway resulted in decreased biological fitness and reduced tolerance to phenazines in comparison with the wild-type strain 8004. These results imply that the tolerance mechanism of the TAT pathway to phenazines is related to the CCM system, but not due to the ROS scavenging system. Furthermore, respiration-related characteristic tests and peptide analysis suggested that disruption of the TAT complex causes a defect in the cytochrome bc1 complex, which may be involved in the tolerance to phenazines. In summary, this study sheds new light on the critical role of the TAT pathway in influencing the fitness and phenazines tolerance of Xanthomonas species.