Resistant starch appears to have promising effects on hypertension, cardiovascular and enteric illness. The influence of resistant starch on intestinal physiological function has drawn great attention. In this study, we first analyzed the physicochemical characteristics, including the crystalline properties, amylose content, and anti-digestibility among different types of buckwheat-resistant starch. The influence of resistant starch on the physiological functions of the mouse intestinal system, contained defecation, and intestinal microbes were also evaluated. The results showed that the crystalline mold of buckwheat-resistant starch changed from A to B + V after acid hydrolysis treatment (AHT) and autoclaving enzymatic debranching treatment (AEDT). The amylose content in AEDT was higher than in AHT and raw buckwheat. Moreover, the anti-digestibility of AEDT was also stronger than that in AHT and raw buckwheat. The buckwheat-resistant starch can promote bowel intestinal tract movement. The quantity of intestinal microbe was regulated by buckwheat-resistant starch. Our research demonstrates an effective preparation method for improving the quality of buckwheat-resistant starch and found that buckwheat-resistant starch has the role of adjusting the distribution of the intestinal flora and maintaining the health of the body.