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Type VI secretion system 2 (T6SS2) of Vibrio parahaemolyticus is required for cell adhesion and autophagy in macrophages; however, other phenotypes conferred by this T6SS have not been thoroughly investigated. We deleted TssL2, a key component of T6SS2 assembly, to explore the role of the T6SS2 in environmental adaptation and virulence. TssL2 deletion reduced Hcp2 secretion, suggesting that TssL2 played an important role in activity of functional T6SS2. We found that TssL2 was necessary for cell aggregation, wrinkly phenotype formation, and participates in motility and biofilm formation by regulating related genes, suggesting that TssL2 was essential for V. parahaemolyticus to adapt changing environments. In addition, this study demonstrated TssL2 significantly affected adhesion, cytotoxicity, bacterial colonization ability, and mortality in mice, even the levels of the proinflammatory cytokines IL-6 and IL-8, suggesting that TssL2 was involved in bacterial virulence and immunity. Proteome analysis revealed that TssL2 significantly affected the expression of 163 proteins related to ABC transporter systems, flagellar assembly, biofilm formation, and multiple microbial metabolism pathways, some of which supported the effect of TssL2 on the different phenotypes of V. parahaemolyticus. Among them, the decreased expression of the T3SS1 and T2SS proteins was confirmed by the results of gene transcription, which may be the main reason for the decrease in cytotoxicity. Altogether, these findings further our understanding of T6SS2 components on environmental adaption and virulence during bacterial infection. KEY POINTS: • The role of T6SS2 in V. parahaemolyticus was far from clear. • TssL2 participates in cell aggregation, wrinkly phenotype formation, motility, and biofilm formation. • TssL2 is essential for cell bacterial colonization, cytotoxicity, virulence, and proinflammatory cytokine production., Competing Interests: Declarations. Ethics approval: All applicable international, national, and institutional animal care and use guidelines were followed. The animal experiments were approved by the Animal Ethics Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (no. SYXK < HU > 2020–0027). Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s).)