A healthy gastrointestinal tract functions as a highly selective barrier, allowing the absorption of nutrients and metabolites while preventing gut bacteria and other xenobiotic compounds from entering host circulation and tissues. The intestinal epithelium and intestinal mucus provide a physical first line of defense against resident microbes, pathogens and xenotoxic compounds. Prior studies have indicated that the gut microbe , a mucin-metabolizer, can stimulate intestinal mucin thickness to improve gut barrier integrity. Grape polyphenol (GP) extracts rich in B-type proanthocyanidin (PAC) compounds have been found to increase the relative abundance of , suggesting that PACs alter the gut microbiota to support a healthy gut barrier. To further investigate the effect of GPs on the gut barrier and , male C57BL/6 mice were fed a high-fat diet (HFD) or low-fat diet (LFD) with or without 1% GPs (HFD-GP, LFD-GP) for 12 weeks. Compared to the mice fed unsupplemented diets, GP-supplemented mice showed increased relative abundance of fecal and cecal , a reduction in total bacteria, a diminished colon mucus layer and increased fecal mucus content. GP supplementation also reduced the presence of goblet cells regardless of dietary fat. Compared to the HFD group, ileal gene expression of lipopolysaccharide (LPS)-binding protein (), an acute-phase protein that promotes pro-inflammatory cytokine expression, was reduced in the HFD-GP group, suggesting reduced LPS in circulation. Despite depletion of the colonic mucus layer, markers of inflammation () were similar among the four groups, with the exception that ileal mRNA levels were lower in the LFD-GP group compared to the LFD group. Our findings suggest that the GP-induced increase in promotes redistribution of the intestinal mucus layer to the intestinal lumen, and that the GP-induced decrease in total bacteria results in a less inflammatory intestinal milieu.