The RNA chaperone Hfq has a multifaceted role in .

is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of in remains unexplored. This study aimed to elucidate the role of in by creating an mutant (Δ) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus . The deletion of resulted in a significantly reduced growth rate during the late exponential phase. Additionally, Δ displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, Δ demonstrated impaired growth, and we observed elevated expression when subjected to and stress conditions. Δ exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the Δ vaccination completely protected catfish against subsequent WT infection. In summary, these results underscore the pivotal role of in , affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.