Human campylobacterosis is one of the most commonly occurring types of bacterial food poisoning in the United States and other developed countries. Most human cases are due to Campylobacter jejuni that is commonly found in the gastrointestinal tract of chickens. The twin-arginine translocase (TAT) secretion system uses N-terminal peptide tags with a distinct twin-arginine-containing motif to identify partially or fully folded proteins and directs them across the cytoplasmic membrane. In other bacteria, the TAT system contributes to diverse phenotypes, including virulence, but the role of this secretion system in Campylobacter pathophysiology is still not well defined. Genome sequence of C. jejuni revealed TAT pathway genes as well as several proteins that contain TAT pathway targeting motifs. The predicted Tat substrates are highly conserved among all sequenced C. jejuni strains. Phenotypic analyses revealed that the tatC knockout has defects in biofilm formation, motility, and flagellation, as well as an increased susceptibility to antimicrobials. Additionally, the tatC mutant was defective in survival under osmotic shock, oxidative, and nutrient stresses. Our results also indicated that tatC is essential for C. jejuni to sustain colonization in chickens. These findings suggest that the TAT pathway affects Campylobacter physiology and contributes to stress responses, allowing this fastidious pathogen to adapt to various environmental conditions.