Abstract: Objective: To study the effect of special milk powder for gastrointestinal tract conditioning on intestinal bowel and intestinal microflora. Methods: After the preparation of special milk powder for gastrointestinal tract conditioning, KM mice were used as the experimental object to establish constipation models with loperamide hydrochloride. The general state, intestinal propulsion rate, defecation situation, and observation of intestinal tissue were used as the investigation indicators, and 16S rRNA gene sequencing was used to analyze the feces of mice to investigate its effects on the bowel movement and intestinal flora of mice. Results: Each dose of special milk powder for gastrointestinal tract conditioning could shorten the time of first defecation in mice (P<0.05) and improve the small intestine thrust rate (P<0.05), but did not damage the goblet cell structure of small intestine in mice. With the increasing of dose, the gastrointestinal conditioning effect of milk powder was better. Although the gastrointestinal tract conditioning special milk powder did not significantly change the richness and evenness of intestinal microbes, it could significantly change the composition of intestinal microecological structure of mice, and significantly increase the relative abundance of some probiotics, Lactobacillus and Bifidobacterium. Conclusion: Special milk powder for gastrointestinal tract conditioning could affect intestinal movement and regulate intestinal flora structure to improve constipation induce by loperamide.