The effect of diets containing different proportions of buckwheat on intestinal function of cholesterol absorption and excretion and intestinal microbiota in hypercholesterolemic mice were studied. The mice were divided into four groups where they were fed with AIN-93M standard diet (control group), a high-cholesterol diet (model group), a high-cholesterol diet + low-dose of buckwheat diet, and a high-cholesterol diet + high-dose of buckwheat diet, respectively. The results showed that diets containing different proportions of buckwheat can all repaired the damaged structure of small intestine to certain extent. Compared with the model group, diets containing different proportions of buckwheat increased the mRNA expression levels of ATP binding cassette transporter subfamily G members 5/8 (ABCG5/ABCG8), and reduced the mRNA expression level of Niemann-Pick type C 1, such as 1 (NPC1L1) protein, in the small intestine, which in turn reduced the absorption of cholesterol in the small intestine. Diets containing different proportions of buckwheat significantly increased the expression of mouse intestinal ABCA1 and SR-B1 genes expression level, which promoted reverse cholesterol transport in the intestine. Compared to the model group, the diet containing a high proportion of buckwheat greatly promoted the abundance of the intestinal microbiota. On the contrary, the diet containing a low proportion of buckwheat had a negative effect on the abundance of the intestinal microbiota.