The sodium-hydrogen exchanger isoform 3 (NHE3) is abundantly expressed in the gastrointestinal tract and plays an essential role in sodium/fluid absorption and acid-base homeostasis. However, understanding the precise role of intestinal NHE3 has been challenging due to the lack of a suitable animal model. To circumvent this problem, we generated a tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout mouse model (NHE3IEC-KO). Before tamoxifen administration, the phenotype and blood parameters of NHE3IEC-KO were unremarkable compared with control mice. After tamoxifen administration, NHE3IEC-KO mice have undetectable levels of NHE3 in the intestine. NHE3IEC-KO mice develop watery, alkaline diarrhea in combination with a dilated small intestine, cecum and colon. They have higher fluid intake compared to controls due to persistent diarrhea. The mortality rate in NHE3IEC-KO is ∼25% after 3 weeks. We found that NHE3IEC-KO mice exhibit metabolic acidosis, lower blood bicarbonate levels, hyponatremia and hyperkalemia associated with drastically elevated plasma aldosterone levels. These results demonstrate that intestinal NHE3 has a significant contribution to acid-base, Na+ and volume homeostasis, and lack of intestinal NHE3 has consequences on intestinal structural integrity. Based on the physiological role of NHE3, pharmacological inhibition of intestinal NHE3 is an interesting treatment strategy. In fact, nonabsorbable NHE3 inhibitors have been developed, and preclinical as well as clinical trials indicate possible pharmacological use in fluid overload, hypertension, chronic kidney disease, hyperphosphatemia, and constipation.