The effect of water-soluble tartary buckwheat protein on the growth and bile salt tolerance of probiotics (Lactobacillus plantarum and L. acidophilus) was explored in an in vitro intestinal environment. The results demonstrated that the addition of different concentrations of tartary buckwheat protein (1, 3 and 5 mg/mL) increased the optical density value (OD600) of L. plantarum culture broth by 12.33%, 25.84% and 35.31%, respectively, and that of L. acidophilus culture liquid by 15.37%, 26.66% and 30.57% respectively. In addition, tartary buckwheat protein increased the tolerance of L. plantarum and L. acidophilus to bile salts (sodium cholate and sodium glycyl cholate) significantly (p<0.05). The OD600 values of L. plantarum culture broth increased 1.64 and 8.67 times and that of L. acidophilus culture broth increased 4.11 and 8.30 times in sodium cholate and sodium glycyl cholate, compared with that of control, respectively. Further in vitro culture experiments demonstrated that the effect of buckwheat protein significantly promoted the growth of intestinal Lactobacilli (p<0.05). In conclusion, tartary buckwheat protein improves the growth of Lactobacillus in bile acid salts and the intestine, indicating scope for the application of tartary buckwheat protein in food.