Aeromonas veronii is an important pathogen in the co-occurrence of humans and fish, causing significant mortality in various aquatic organisms, posing a major threat to aquaculture and public health care. The type Ⅲ secretion system (T3SS) is one of the most critical virulence mechanisms in determining the pathogenicity of Aeromonas species. The transcription factor ExsA of AraC family serves as the main regulatory factor and activates T3SS assembly and effector protein secretion in response to changes in environmental conditions. In order to explore the effect of exsA on the pathogenicity of the A. veronii, this study constructed an exsA gene knockout strain using homologous recombination strategy and tested its biofilm formation, adhesion capabilities, as well as the cytotoxicity of the spent cultural supernatant. The results showed that the deletion of the exsA gene in A. veronii led to a significant decrease of its biofilm formation capability, its adhesion to ex vivo host tissues, and the cytotoxicity of the spent culture supernatants. Compared with the wild type, the ability of the exsA knockout strain to form biofilm was reduced by 1.5 folds. The adhesion to epithelial cells was not significantly changed in exsA knockout strain as compared with the wild type, while the adhesion to ex vivo mouse intestinal tissues decreased by 3.4 folds and the cytotoxicity of bacterial culture supernatants decreases by 1.2 folds. These results proved that exsA plays an important regulatory role in the virulence mechanism of bacterial pathogen, laying a solid foundation for further research on the function of T3SS in the pathogenic mechanism of A. veronii and provided clues for establishing strategies for the control of the aquatic infection.