The newest member of the Na⁺/H⁺ exchanger (NHE) family, NHE8 is abundantly expressed at the apical membrane of the intestinal epithelia. We previously reported that Muc2 expression was significantly decreased in the colon in NHE8^-/- mice, suggesting NHE8 is involved in intestinal mucosal protection. In this study, we further evaluated the role of NHE8 in intestinal epithelial protection after DSS challenge. Compared with wild-type mice, NHE8^-/- mice have increased bacterial adhesion and inflammation, especially in the distal colon. NHE8^-/- mice are also susceptible to DSS treatment. Real-time PCR detected a remarkable increase in the expression of IL-1β, IL-6, TNFα and IL-4 in DSS-treated NHE8^-/- mice compared with DSS-treated wild-type littermates. Immunohistochemistry showed a disorganized epithelial layer in the colon of NHE8^-/- mice. Periodic Acid-Schiff staining showed a reduction in the number of mature goblet cells and the area of the goblet cell theca in NHE8^-/- mice. Phyloxine/tartrazine staining revealed a decrease in functional Paneth cell population in the NHE8^-/- small intestinal crypt. The expression of enteric defensins was also decreased in NHE8^-/- mice. The reduced mucin production in goblet cells and antimicrobial peptides production in Paneth cells lead to disruption of the intestinal mucosa protection. Therefore, NHE8 may be involved in the establishment of intestinal mucosal integrity by regulating the functions of goblet and Paneth cells.