Aim SLC26A3 (DRA) mediates the absorption of luminal Cl(-)in exchange for HCO(3)(-)in the distal intestine. Its expression is lost in congenital chloride diarrhoea (CLD) and strongly decreased in the presence of intestinal inflammation. To characterize the consequences of a loss of Slc26a3 beyond disturbed electrolyte transport, colonic mucus synthesis, surface accumulation and composition, pH microclimate, microbiome composition and development of inflammation was studied inslc26a3(-/-)mice. Methods The epithelial surface pH microclimate and the surface mucus accumulation in vivo was assessed by two photon microscopy in exteriorized mid colon of anaesthetizedslc26a3(-/-)andwtlittermates. Mucus synthesis, composition and inflammatory markers were studied by qPCR and immunohistochemistry and microbiome composition by 16S rRNA sequencing. Results Colonic pH microclimate was significantly more acidic inslc26a3(-/-)and to a lesser extent incftr(-/-)thanin wtmice. Goblet cell thecae per crypt were decreased inslc26a3(-/-)and increased incftr(-/-)colon. Mucus accumulation in vivo was reduced, but much less so than incftr(-/-)colon, which is possibly related to the different colonic fluid balance.Slc26a3(-/-)colonic luminal microbiome displayed strong decrease in diversity. These alterations preceded and maybe causally related to increased mucosalTNF alpha mRNA expression levels and leucocyte infiltration in the mid-distal colon ofslc26a3(-/-)but not ofcftr(-/-)mice. Conclusions These findings may explain the strong increase in the susceptibility ofslc26a3(-/-)mice to DSS damage, and offer insight into the mechanisms leading to an increased incidence of intestinal inflammation in CLD patients.