Sodium/hydrogen exchangers (NHEs) play a major role in Na ϩ absorption, cell volume regulation, and intracellular pH regulation. Of the nine identified mammalian NHEs, three (NHE2, NHE3, and NHE8) are localized on the apical membrane of epithelial cells in the small intestine and the kidney. Although the regulation of NHE2 and NHE3 expression has been extensively studied in the past decade, little is known about the regulation of NHE8 gene expression under physiological conditions. The current studies were performed to explore the role of epidermal growth factor (EGF) on NHE8 expression during intestinal maturation. Brush-border membrane vesicles (BBMV) were isolated from intestinal epithelia, and Western blot analysis was performed to determine NHE8 protein expression of sucking male rats treated with EGF. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcriptional factors involved in EGF-mediated regulation. Our results showed that intestinal NHE8 mRNA expression was decreased in EGF-treated rats and Caco-2 cells, and NHE8 protein abundance was also decreased in EGF-treated rats. The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by EGF treatment. This could be explained by reduced binding of transcription factor Sp3 on the NHE8 basal promoter region in the presence of EGF. Pretreatment with MEK1/2 inhibitor UO-126 could prevent EGF-mediated inhibition of NHE8 gene expression. In conclusion, this study showed that EGF inhibits NHE8 gene expression through reducing its basal transcription, suggesting an important role of EGF in regulating NHE expression during intestinal maturation. intestine; Sp3; sodium/hydrogen exchangers; Caco-2 cells SODIUM IS AN ELECTROLYTE that is essential to volume regulation and maintenance of blood pressure. Sodium absorption is mediated by several transporter families including Na . They are widely expressed in mammalian cells, with broad physiological functions including intracellular pH homeostasis, cell volume regulation, acid-base regulation, and electroneutral NaCl transport. To date, nine mammalian NHEs are discovered, and five of them (NHE1-4, 8) are located in intestinal enterocytes (36,40). These NHEs have different membrane localization and functions. NHE1 and NHE4 are expressed in the basolateral membrane of the intestinal epithelial cells (26,30,32), whereas NHE2, NHE3, and NHE8 are expressed in the brush-border membrane (BBM) of the intestinal epithelial cells (10,12,36). NHE1 contributes to cell volume regulation and intracellular pH (pH i ) regulation (26). Lack of NHE1 expression results in decreased postnatal growth rate, ataxia, and seizures (4). NHE2 is involved in gastric function. Loss of NHE2 results in altered oxyntic mucosa, markedly reduced parietal and zymogenic cell number (5, 21), and impaired intestinal barr...