AbstractLuxR‐type transcriptional factors are essential in many bacterial physiological processes. However, there have been no reports on their roles in Aeromonas hydrophila. In this study, six stable silent strains were constructed using shRNA. Significant decreases in the expression levels of luxR05, luxR08, luxR19, luxR11, luxR164 and luxR165 were shown in their respective strains by qRT‐PCR. The luxR05‐RNAi and luxR164‐RNAi exhibit the most significant changes in sensitivity to kanamycin and gentamicin. The luxR05‐RNAi showed minimum biofilm formation and the least motility, while luxR164‐RNAi showed minimum biofilm formation, adhesion, growth and extracellular protease activity compared to the wild‐type strain. In summary, the results of this paper suggest that all six luxR genes are involved in multiple physiological processes in A. hydrophila and that the roles of luxR05 and luxR164 are highly significant. The sensitivity of luxR05‐RNAi and luxR164‐RNAi to drugs may be closely related to biofilm formation. The luxR05 may play an important role in the pathogenicity of A. hydrophila by regulating the movement, adhesion and biofilm formation of bacteria, whereas luxR164 may be involved in similar functions by regulating bacterial adhesion, extracellular enzyme activity and growth. These results help further our understanding of the drug resistance and pathogenesis of A. hydrophila.