Diarrhea of piglets is an important disease affecting the development of pig industry, while F4 enterotoxigenic Escherichia coli (ETEC) is an important pathogen causing diarrhea of newborn or weaned piglets, which seriously harms animal husbandry and causes huge economic losses. The pathogenicity of E. coli is closely related to its virulence factors, which are strictly regulated in vivo, and the quorum sensing system is involved in this process. To study the effect of quorum sensing signal molecule acyl homoserine lactone (AHL) on the biological characteristics of Enterotoxigenic E. coli (ETEC), gene lasI of Pseudomonas aeruginosa which is responsible for the synthesis of long side-chain AHL (3OC12-HSL), and gene yenI of Yersinia enterocolitica which is responsible for the synthesis of short side-chain AHL (C6-HSL), were selected and transformed into ETEC strain C83902, respectively, and endowed with the ability of endogenous synthesis of 3OC12-HSL and C6-HSL. At the same time, 200 μmol/L 3OC12-HSL and C6-HSL were added into the culture medium, respectively, and co-cultured with ETEC strain C83902. ETEC strains were stimulated by exogenous and endogenous long-chain and short-chain AHL molecules, respectively, and their biological characteristics, such as growth characteristics, biofilm formation ability, and adhesion ability to piglets intestinal epithelial cells IPEC-J2 were observed under the influence of the AHLs described above. Real-time PCR was used to detect the transcription levels of genes encoding major subunits of pathogenic factors such as fimbriae, flagella, enterotoxin, adhesin and hemolysin under the influence of the AHLs. The results showed that under the influence of quorum sensing AHLs, the biofilm formation ability and adherence ability of ETEC were significantly reduced, and the expression of fliC and other major virulence factors were regulated. AHL signaling molecules can regulate virulence related characteristics of F4ac+ ETEC.