Abstract: In this paper, water-soluble buckwheat protein was used as the research object, and the p H and the quantity of short-chain fatty acids ( acetic acid, propionic acid, butyric acid, lactic acid) and harmful bacteria ( Enterococcus, Enterobacteriaceae and Clostridium perfringens) were determined by simulated in vitro fermentation.The mechanism of buckwheat protein inhibition of intestinal harmful flora growth was investigated.The results showed that the addition of different concentrations of buckwheat protein could reduce the number of harmful bacteria compared with the control group.The high dose group reduced the quantity of Enterococcus, Enterobacter and Clostridium perfringens by 65%, 72% and 78% ( 32 h) . The tartary buckwheat protein in different dosage groups decreased the p H of the fecal fermentation broth by 35%, 39% and 40% ( 32 h) , respectively.In addition, the concentration of acetic acid, propionic acid, butyric acid and lactic acid in high dose group were increased by8.94, 33.77, 27.34 and 25.55 times ( 32 h) , respectively, compared with the control group.In summary, tartary buckwheat protein can improve the intestinal short-chain fatty acid content, thereby reducing the intestinal environment p H, inhibit intestinal harmful bacteria ( Enterococci, Enterobacteriaceae, Clostridium perfringens) growth.