Chronic inflammation and enteric infections are frequently associated with epithelial Na⁺/H⁺ exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3^-/- mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3-deficiency mediated colitis. We herein characterized the colonic microbiome of wildtype (WT) and NHE3^-/- mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3^-/- mice as compared to WT. Rederivation of NHE3^-/- mice from conventional to an ultraclean barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3^-/- mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared to reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3^-/- mice and reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. Therefore, NHE3 deficiency may be a critical contributor to dysbiosis observed in IBD patients.