Aeromonas hydrophila is a common pathogen in water environment and has strong pathogenicity. In this study, the gene aguA of A. hydrophila was knocked out for the first time. Transcriptome analysis and qRT-PCR indicated that the knockout of aguA down-regulated the bacterial adhesion process, pathogenic process, amine metabolism process and etc. In addition, type Ⅵ secretion system genes, type Ⅱ secretion system genes, flagella synthesis-related genes, fimbriae synthesis-related genes, hemolysis genes, type Ⅰ secretion system genes, enterotoxin genes, and RTX genes were also down-regulated at the transcriptome level. qRT-PCR showed that most of these genes were significantly down-regulated. Compared with the wild strain, the pathogenicity of A. hydrophila aguA-knocked-out strain infected grass goldfish was reduced by 86.6% through infection experiment in vivo, and it was found that the biofilm formation ability of the aguA-knocked-out strain was reduced by 16.6%. The above results show that aguA gene is closely related to the pathogenicity of A. hydrophila.