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Potassium diformate (KDF) is a kind of formate, which possesses the advantages of antimicrobial activity, growth promotion and preventing diarrhea in weaned piglets. However, the researches of KDF in animal production mostly focused on apparent indexes such as growth performance and the mechanisms of KDF on intestinal health have not been reported. Thus, porcine small intestinal epithelial cells (IPEC-J2) infected with Enterohemorrhagic Escherichia coli (EHEC) was used to investigate the role of KDF on alleviating intestinal inflammation in this study. The 0.125 mg/mL KDF treated IPEC-J2 cells for 6 h and IPEC-J2 cells challenged with 5 × 107 CFU/mL EHEC for 4 h were confirmed as the optimum concentration and time for the following experiment. The subsequent experiment was divided into four groups: control group (CON), EHEC group, KDF group, KDF+EHEC group. The results showed that KDF increased the cell viability and the gene expression levels of SGLT3 and TGF-β , while decreased the content of IL-1β compared with the CON group. The cell viability and the gene expressions of SGLT1 , SGLT3 , GLUT2 , Claudin-1 , Occludin and TGF-β , and the protein expression of ZO-1 in EHEC group were lower than those in CON group, whereas the gene expressions of IL-1β , TNF , IL-8 and TLR4 , and the level of phosphorylation NF-кB protein were increased. Pretreatment with KDF reduced the content of IgM and IL-1β, the gene expressions of IL-1β , TNF , IL-8 and TLR4 and the level of phosphorylation NF-кB protein, and increased the gene expression of TGF-β and the protein expression of Occludin in IPEC-J2 cells infected EHEC. In conclusion, 0.125 mg/mL KDF on IPEC-J2 cells for 6 h had the beneficial effects on ameliorating the intestinal inflammation because of reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines through regulating NF-кB signaling pathway under the EHEC challenge. • The mechanism of potassium diformate (KDF) alleviating intestinal injury caused by EHEC was elucidated. [ABSTRACT FROM AUTHOR]