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Vibrio anguillarum is a pathogen responsible for vibriosis in aquaculture animals. The formation of bacterial biofilm contributes to infections and increases resistance to antibiotics. Tryptophanase and its substrate tryptophan have been recognized as signal molecules regulating bacterial biofilm formation. This study aimed to investigate the role of the trpA gene encoding tryptophan synthase in V. anguillarum through constructing a trpA mutant (ΔtrpA) and its complemented strain (CΔtrpA). The ΔtrpA produced less tryptophan compared to the WT and ΔtrpA strains, with almost no detectable indole synthesis in the ΔtrpA mutant. RNAseq analysis showed that 152 genes were differentially expressed in the ΔtrpA mutant, including 64 upregulated and 88 downregulated genes. KEGG enrichment analysis and qRT-PCR validation indicated that genes associated with bacterial chemotaxis, two component system, quorum sensing and biofilm formation were downregulated. Crystal violet staining confirmed that the ΔtrpA mutant decreased biofilm formation due to the reduced tryptophan and indole. Our studies also showed that TrpA plays an important role in the motility and hemolytic ability of V. anguillarum. Finally, the virulence of the ΔtrpA mutant was evaluated using a Tetrahymena infection model, which demonstrated that the virulence of ΔtrpA mutant was significantly attenuated. These findings provide insight into the role of trpA gene in biofilm formation, motility, hemolysis, and virulence in V. anguillarum., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Ltd.)