Na⁺/H⁺ exchanger 3 (NHE3) provides a major route for intestinal Na⁺ absorption. It has been considered a target of proinflammatory cytokines and enteropathogenic bacteria and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3^-/- mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse and reduced body weight. Genome-wide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of BrdU-positive cells; diffuse neutrophilic infiltrate with concomitant 15-fold increase in MMP-8 expression; an increased number of pSer²⁷⁶-RelA-positive cells and a significant decrease in PAS-positive goblet cells. Real-time PCR demonstrated elevated expression of iNOS (38-fold), TNF (6-fold), MIP-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3^-/- mice. NHE3^-/- mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis, may modulate the extent and severity of colonic inflammation.