To explore the regulatory function of the twocomponent system (TCS) in Aeromonas hydrophila,envZ and ompR genes were knocked out via homologous recombination. In vitro,the growth of each strain under different salt concentration stress was tested,the biofilm formation capacity of each strain was compared via the crystal violet staining test,and the resistance to fish blood killing was examined via whole blood killing assay. In vivo,LD50 of each strain was calculated by infecting zebrafish,the systemic invasive ability of each strain in grass carp was compared,and the transcription levels of proinflammatory cytokines were measured by quantitative PCR. The results showed that the ΔenvZ/ompRgrowth did not show significant differences at different salt concentrations in vitro. However,with the deletion of envZ/ompR,the transcription levels of the downstream genes ompF and ompC were significantly affected. ΔenvZ/ompR also showed a significant decrease in biofilm formation and resistance to fish whole blood killing compared with the wild type. In the zebrafish scratch infection model,the LD50 of ΔenvZ/ompRincreased 4.8fold compared to the wild type,indicating reduced virulence. In the ΔenvZ/ompRinfected grass carp,the systemic spreading ability of ΔenvZ/ompRwas significantly weaken,and the transcript levels of pro-inflammatory factors TNF-αand IL-6 were significantly reduced in the spleen,head-kidney,and kidney. Taken together,the above results indicated that the TCS EnvZ/OmpR involved in response to osmotic stress,in the regulation of biofilm formation and in pathogenic processes,especially in the systemic diffusion process in the host.