The bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins and plays pleiotropic roles in growth, motility, and the secretion of some virulent factors. In this study, the authors identified the Tat gene cluster in fish pathogen Vibrio alginolyticus and explored its roles in pathogenesis toward fish. Vibrio alginolyticus Tat mutants showed growth deficiency in TMAO medium, while the complement strain restored the ability to grow in the medium, demonstrating the conservative function of the Tat system in translocation of redox enzymes or cofactors in this bacterium. In V. alginolyticus, deletion of the tatABC genes led to a drastic decrease in biofilm biogenesis. Interestingly, the secretion of extracellular protease Asp, an established exotoxin of the bacterium, was significantly decreased in the TatC mutant, suggesting that TatC might play a part in the production of virulence factors in the bacterium. Furthermore, the Tat mutants displayed attenuated virulence toward the fish model and EPC cells. These findings suggest that the Tat secretion related to the extracellular protease activity as well as virulence in V. alginolyticus provided new insights into the pathogenesis of vibriosis in fish.