Vibrio alginolyticus, a Gram-negative bacterium, is an opportunistic pathogen of both marine animals and humans, resulting in significant losses in the aquaculture industry. Type III secretion system (T3SS) is a crucial virulence mechanism ofV. alginolyticus. In this study, the T3SS regulatory geneexsA, which was cloned fromV. alginolyticuswild-type strain HY9901, is 861 bp encoding a protein of 286 amino acids. The ΔexsAwas constructed by homologous recombination and Overlap-PCR. Although there was no difference in growth between HY9901 and ΔexsA, the ΔexsAexhibited significantly decreased extracellular protease activity and biofilm formation. Besides, the ΔexsAshowed a weakened swarming phenotype and an ~100-fold decrease in virulence to zebrafish. Antibiotic susceptibility testing showed the HY9901ΔexsAwas more sensitive to kanamycin, minocycline, tetracycline, gentamicin, doxycycline and neomycin. Compared to HY9901 there were 541 up-regulated genes and 663 down-regulated genes in ΔexsA, screened by transcriptome sequencing. qRT-PCR and β-galactosidase reporter assays were used to analyze the transcription levels ofhopgene revealing thatexsAgene could facilitate the expression ofhopgene. Finally,Danio rerio, vaccinated with ΔexsAthrough intramuscular injection, induced a relative percent survival (RPS) value of 66.7% after challenging with HY9901 wild type strain. qRT-PCR assays showed that vaccination with ΔexsAincreased the expression of immune-related genes, includingGATA-1, IL6, IgM, andTNF-α in zebrafish. In summary, these results demonstrate the importance ofexsAinV. alginolyticusand provide a basis for further investigations into the virulence and infection mechanism.