Objective: To investigate sRNA to regulate the intracellular survival and virulence of Edwardsiella tarda (Et). Methods: In this study,the analysis of ET13 sRNA was carried out according to the sequence of transcripts and bioinformatics methods. The transcription levels of sRNAs under different stress conditions (low acid,nutrition deficiency,oxidation and high salt) were detected by RT-PCR,and the high expression of sRNA was screened out. The length and transcription initiation and termination sites of EsR240 were found by Northern blotting and RACE assay. BLAST was used to analyze the conservatism of EsR240. IntaRNA software was used to predict EsR240 target genes,and qRT-PCR was used to detect the expression levels of these target genes. The EsR240 deletion strain and complementary strain of ET13 were constructed by suicide plasmid homologous recombination. The survival rate,intracellular viability and virulence of type ET13,its EsR240 deletion strain and complementary strain under different extracellular stress conditions were compared,as well as their ability to survive in macrophages and their virulence to mice. Results: According to the results of RNA-sequencing of ET13 strain,it was compared with the annotated gene of protein library,the candidate sRNA of non-alignment was compared with the sRNA library. 13 new sRNA,RT-PCR with promoters and ρ-independent terminators were found by software,showing that 8 of them had different transcription levels under various stress conditions,and EsR128,EsR139 and EsR240 transcription levels were relatively higher. Northern blotting confirmed that EsR240 was transcribed during the growth and stable stages of Et strain,with a size of 596 bp. The transcription initiation and termination sites of EsR240 were determined by RACE assay. BLAST analysis showed that EsR240 was common in Et strains,which was consistent with the characteristics of sRNA. IntaRNA was used to predict EsR240 target gene by software. qRT-PCR results showed that EsR240 regulated most of the predicted target gene expression. These target genes may be the adenosine triphosphate binding cassette (ATP binding cassette,ABC)-F transporter,the regulator of FtsH protease YccA,Na⁺/H⁺ reverse transporter and glycoside hydrolase,etc. Under different conditions,the number of target genes regulated by EsR240 was different. The deletion of EsR240 significantly affected the survival rate and intracellular viability of Et under different extracellular stress conditions,and affected the toxicity of Et to mice. Conclusion: EsR240 affects the intracellular survival of Et by regulating the target gene,which is a sRNA that regulates the virulence of Et to mice.