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Aquaculture plays important roles in global food security. Prized as a valuable commercial species, the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis) has faced unprecedented challenge due to bacterial diseases, including vibriosis caused by Vibrio anguillarum. Alternative splicing (AS) is an important layer of post-transcriptional regulation, being involved in almost all biological processes, including development, reproduction, and immunity. However, its roles in immunity in half-smooth tongue sole remain elusive. In this study, therefore, we profiled the changes of alternative splicing in half-smooth tongue sole after infection with different concentrations of V. anguillarum , and investigated their functions by both enrichment analysis and targeted gene transcript overexpression analysis with half-smooth tongue sole macrophages. Intriguingly, differentially alternatively spliced (DAS) genes were found to be involved in RNA splicing for each bacterial treatment and for the shared gene set, consistent with previous studies that splicing factor themselves often underwent alternative splicing. A set of 12 immune-related shared DAS genes were identified, including hp , c5 , and traf2b. Targeted gene analysis using the shared DAS gene, haptoglobin (hp), showed that alternative splicing resulted in changes of protein domains, and overexpression analysis demonstrated that two transcripts of hp exhibited contrary roles in stimulating or reducing the expression of immune factors in response to lipopolysaccharides (LPS), reflecting the vital roles of alternative splicing in immunity of tongue sole. DAS events were further verified by qRT-PCR (20 primer pairs for 10 DAS events) to confirm the reliability of our bioinformatics analysis. This study, for the first time, revealed the involvement of alternative splicing in immune responses of half-smooth tongue sole using both omics data and targeted gene functional analysis, setting a solid foundation for further analysis and providing clues for potential usage in controlling bacterial diseases in aquaculture. • RNA splicing was enriched for DAS genes in each comparison and the shared gene set. • A set of 12 immune-related genes were identified in 104 shared DAS genes. • Two hp splicing isoforms exhibited contrary roles in stimulation of immune factors. [ABSTRACT FROM AUTHOR]