Background: Patients with congenital chloride diarrhea (CLD), caused by loss of function mutations of SLC26a3 (DRA), display a propensity for acute and chronic intestinal inflammation. Conversely, patients with intestinal inflammation, as well as murine inflamed colon, display a defect in surface cell anion exchange, luminal alkalinisation rates, and a decrease in DRA mRNA and protein expression. Aim: In this study, we investigated the effect of genetic ablation of Slc26a3 (DRA) on the surface pH and the dynamics of mucus layer buildup in vivo by two photon microscopy, on the intestinal microbiome by 16S rRNA sequencing, and on the inflammatory state of the mucosa by qPCR and immunohistochemistry, under nonchallenged, specific pathogen-free conditions. Methods and Results: DRA-deficient mice, an animal model for congenital Cl- diarrhea (CLD), and WT littermates were anesthetized by 1.5-2% isoflurane-inhalation anesthesia and the intestinal surface pH was assayed by two photon microscopy in exteriorized vascularly perfused colon using SNARF1 free acid. DRA-deficient mice displayed strongly abnormal low surface pH and loss of fluid absorption in the mid-distal colon, but a virtually normal mucus layer buildup rate. However, they lacked a firmly adherent mucus layer. In addition, they displayed altered microbiota composition with a decrease in the firmicutes and an increase in the bacteroidetes in all parts of their colon. However, only the distal colon, which displays the thickest mucus layer and the highest DRA expression levels in WT mice, showed evidence for inflammation in the DRA KO mice, indicated by an increase in infiltrating mononuclear cells and increased TNF-alpha expression. Conclusions: Apart from absorbing Cl-, the anion exchanger Scl26a3 (DRA) is intimately involved in the maintenance of intestinal barrier properties such as high colonic surface pH, firmly adherent mucus layer, and the composition of the microbiome. Because the microbiota composition was different from WT in all parts of the DRA-deficient colon, but inflammation was seen only in the mid-distal colon, we speculate that the reason for inflammation is a combination of weakened barrier properties with a more aggressive microbiota in the absence of DRA expression. Intestinal inflammation is thus both a consequence of, and a cause for, disregulated ileocolonic HCO3- transport.