Abstract: Aeromonas veronii, a gram-negative pathogenic bacterium, causes a variety of diseases in aquatic animals and human, which poses a severe threat to the aquaculture industry and human public health but the pathogenesis mechanism is not clear. The genomic analysis of A.veronii C4 revealed the existence of the gene encoding an unknown AraC family transcription factor, named as asfR by us, located at the adjacent site of the flagellum synthesis gene fliE that is related to the type Ⅲ secretion system of bacteria. Prediction by NCBI CD-Search indicated that AsfR contained a conserved HTH DNA binding domain and a GyrI-like small molecule binding domain. The AraC protein superfamily is known to be ubiquitous in bacteria and is involved in the regulation of various cellular processes, including carbon metabolism, stress responses, bacterial virulence and pathogenicity. AsfR is highly conserved in AVNIH1 and TH0426 strains of A. veronii, but further studies on the biological functions of this protein have not been reported yet. In order to further study the function of the protein, homologous recombination technology was used to construct asfR gene-knockout strains in this study. We also found that asfR deletion did not affect the growth of the bacteria under nutrient-rich circumstance, but significantly increased growth rate under the adversity condition, compared with wild-type strain. Moreover, the abilities of biofilm formation decreased in asfR deletion strain. The results demonstrated that AsfR was involved in the biofilm formation and stress resistance of A. veronii. This study provides a necessary tool and the primary theoretical basis for the further identification of the biological functions of the protein AsfR in A.veronii C4.