Abstract: In order to investigate the function of AraC-family transcriptional factor AetY (U876_01475), we constructed the aetY deletion mutant strain ΔaetY using homologous recombination with Aeromonas hydrophila Chinese epidemic strain NJ-35 as a parental strain. The corresponding gene-complemented strain CΔaetY was also constructed. Biofilm formation ability, auto-aggregation activity, drug resistance to enrofloxacin and pathogenicity were detected in NJ-35, ΔaetY and CΔaetY strains. The inactivation of aetY caused a significant decrease in biofilm formation, auto-aggregation and MIC value for enrofloxacin. The virulence of ΔaetY to Danio rerio was significantly reduced, and the LD₅₀ value had an increase of more than 400 times. The above characteristics of CΔaetY were restored to the same level of the wide type strain. This study for the first time investigated the function of AraC-family transcriptional factor AetY in A. hydrophila, which will provide a basis for further exploration of AetY role in pathogenic mechanism.