The relationship between the intestinal microbiota and the hypocholesterolemic and antiobesity effects of whole grape seed flour from white and red winemaking was evaluated. Male Golden Syrian hamsters were fed a high-fat (HF) control diet or a HF diet supplemented with 10% partially defatted grape seed flours from either Chardonnay (ChrSd) or Cabernet Sauvignon (CabSd) grapes for 3 weeks. The numbers of total bacteria and relative abundances of Bifidobacterium spp., Lactobacillus spp., and Firmicutes in feces were significantly lower, while the relative abundance of Bacteroides fragilis was greater than the control from feeding the ChrSd diet. The ratio of Firmicutes/Bacteroidetes (F/B) was lower in the ChrSd diet. There were significantly positive correlations between Lactobacillus spp., ratio of F/B, and plasma total- and LDL-cholesterol and liver weight. The reduction of Lactobacillus spp. by the ChrSd diet was accompanied by inhibition of Farnesoid X receptor (FXR) signaling in the intestine as expression of intestinal fibrablast growth factor (FGF)15, positively regulated by FXR, was decreased. Expression of CYP7A1, negatively regulated by FGF15, was up-regulated in the liver, which indicates that alteration of the intestinal microbiota may regulate bile acid and lipid metabolism. These findings suggest that beneficial health effects of Chardonnay grape seed flour on HF-induced metabolic disease relate in part to modulation of intestinal microbiota and their metabolic processes.