NHE8 transporter is a member of the sodium-hydrogen exchanger (NHE) family. This transporter protein is expressed at the apical membrane of the epithelial cells of the kidney and intestine, and contributes to the vectorial Na⁺ transport in both tissues. Although NaCl absorption has been shown to be reduced in diarrhea associated with colitis and enteritis, little is known about the role of Na⁺/H⁺ exchange and the involvement of NHE isoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea. This current study investigated the role of NHE8 in the setting of inflammatory states. Jejunal mucosa was harvested from TNBS (trinitrobenzene sulfonic acid) colitis rats or LPS (lipopolysaccharide) rats for RNA extraction and brush-border membrane (BBM) protein purification. The human NHE8 gene promoter was cloned from human genomic DNA and characterized in Caco-2 cells. The promoter was further used to study the mechanisms of TNF- mediated NHE8 expression down-regulation in Caco-2 cells. Results from Western blot and Real-Time PCR indicated that NHE8 protein and mRNA were significantly reduced in TNBS rats and LPS rats. In Caco-2 cells, TNF- produces similar reduction levels in the endogenous NHE8 mRNA expression observed in our in vivo studies. The down-regulation of NHE8 expression mediated by TNF- could be blocked by transcription inhibitor actinomycin D, suggesting the involvement of transcriptional regulation. Further studies indicated that the human NHE8 gene transcription could be activated by Sp3 transcriptional factor, and TNF- inhibits human NHE8 expression by reducing Sp3 interaction at the minimal promoter region of the human NHE8 gene. In conclusion, our studies suggest that TNF- decreases NHE8 expression in inflammation induced by TNBS and LPS, which may contribute to the diarrhea associated with inflammation.