RegIII and RegIII are antimicrobial peptides expressed in intestinal epithelial cells.Expression of these peptides is reportedly decreased by high-fat diet (HFD) and increased by indigestible oligosaccharides in mice. Clearly, these dietary regimens change the structure of intestinal microbiota. We employed an intestinal microbiota transplantation (IMT) to test whether diet-induced changes in the expression of these peptides are mediated by gut microbiota. C57BL/6J mice were fed either a normal-fat diet (NFD), a HFD, or a NFD supplemented with or without 1-kestose (KES), an indigestible oligosaccharide. Ileal RegIII and RegIII mRNA levels were lower in mice receiving IMT from HFD-fed mice than in those receiving NFD-fed mice and higher in mice receiving IMT from KES-supplemented mice than in those receiving the mice without KES supplementation. Western blot analysis showed that serum RegIII levels changed in parallel with the ileal mRNA levels. We propose that HFDand KES-induced changes in the ileal RegIII and RegIII expression and in the circulating RegIIIβ levels are mediated, at least in part, by intestinal microbiota.