To investigate the role of nr4a1 in the defense against Aeromonas hydrophila infection in fish, this study employed various techniques, such as bioinformatics, CRISPR-Cas9 gene editing, and quantitative real-time PCR (qPCR) to analyze the gene structure and expression pattern of nr4a1 in zebrafish (Danio rerio). Subsequently, we established an nr4a1 knockout zebrafish model and conducted in vitro overexpression experiments of nr4a1, revealing its critical role in the anti-infection process. The results showed that the zebrafish nr4a1 gene sequence is highly conserved, with an open reading frame encoding a polypeptide chain of 574 amino acids. The predicted protein has an isoelectric point of 5.79 and a relative molecular mass of 63.04. The zebrafish nr4a1 was widely expressed across various fish tissues. Besides, the nr4a1 mutant zebrafish exhibited a 7 bp deletion in the second exon, resulting in premature termination of translation and a truncated polypeptide chain, reducing its length from 574 to 297 amino acids. Following A. hydrophila infection, the relative expression of the nr4a1 in zebrafish liver tissues initially decreased and then increased. Compared to wild-type controls, nr4a1 knockout zebrafish exhibited a lower survival rate,a significantly higher bacterial loads in liver tissues, and more severe hepatic pathological damage. The expression levels of il-1β, tnf-α, nf-κB and il-22 were significantly elevated in nr4a1 knockout zebrafish compared to their wild-type counterparts. Additionally, overexpression of nr4a1 in the zebrafish ZF4 cell line significantly inhibited the upregulation of inflammatory factors induced by A. hydrophila infection, suggesting that this effect may be mediated through the NF-κB signaling pathway. These results indicate that Nr4a1 can suppress the expression of pro-inflammatory factors, thereby playing a crucial protective role in the immune inflammatory response of zebrafish to A. hydrophila infection.