BACKGROUND: The Highly Pathogenic Island (HPI) found in Yersinia pestis can be horizontally transferred to E. coli, enhancing its virulence and pathogenicity. Ubiquitin (Ub) acts as an activator of the NF-κB pathway and plays a critical role in the inflammatory response. However, the precise mechanism by which Ub and the regulated TLR4/NF-κB pathway contribute to HPI-induced intestinal inflammation in E. coli remains unclear. RESULTS: In this study, we established Ub overexpression models of small intestinal epithelial cells (in vitro) and BALB/c mice (in vivo) and infected these models with HPI-rich E. coli. We investigated the role of the Ub-regulated TLR4/NF-κB pathway in E. coli HPI-induced intestinal inflammation through qPCR, ELISA, immunofluorescence, immunohistochemistry, and H&E staining. Our findings confirmed that E. coli HPI promoted the expression of Ub, TLR4, and NF-κB in IPEC-J2 cells and induced the translocation of NF-κB p65 protein to the nucleus. Further investigations revealed that Ub overexpression enhanced epithelial cell damage induced by E. coli HPI. This was accompanied by up-regulation of mRNA levels of TLR4, MyD88, NF-κB, IL-1β, and TNF-α, as well as increased release of the inflammatory factors IL-1β and TNF-α. In a mouse model with Ub overexpression infected with E. coli HPI, we observed that Ub overexpression promoted E. coli HPI-induced intestinal inflammation. Mechanistically, E. coli HPI induced intestinal epithelial cell damage by inducing Ub overexpression and modulating the TLR4/NF-κB pathway. CONCLUSIONS: In conclusion, this study sheds light on the significant role of the Ub-regulated TLR4/NF-κB pathway in E. coli HPI-induced duodenitis, offering novel insights into the pathogenesis of E. coli infections.