Abstract: The effects of five strains of Lactobacillus paracasei from Tibet on constipation and gut microbiota of mice were studied. 160 male Kunming mice were randomly divided into two groups: A and B. Each group was randomly divided into a blank subgroup, a model subgroup, five experimental strain (including T1-5, T1-7, T1-9, T1-d, and 5B-1) subgroup and a control strain 6108 for the laxative function test, with 10 mice in each subgroup. Among them, 80 mice in group A were used to measure the small intestine propulsion rate of mice, and 80 mice in group B were used to measure defecation status. Strains T1-5, T1-9, and 5B-1 with good performance in laxative function test were then selected to detect the regulation of intestinal microbiota and total 24 male BALB/c mice were selected for this experiment, and were divided into a blank subgroup, a T1-5 subgroup, T1-9 subgroup, and 5B-1 subgroup, with 6 mice in each subgroup. The experimental results of the laxative function test showed that: compared with the model group, the small intestine propulsion rate of mice in T1-9 and 5B-1 groups was significantly higher than that of the model group (P<0.01). The first red stool time was significantly shortened in the T1-9 and 5B-1 groups (P<0.01). The number of fecal particles in the T1-9 group, 5B-1 group, and control strain 6108 group increased significantly at 6 hours (P<0.05). The wet weight of feces in the T1-9 and 6108 groups increased significantly (P<0.05). The experimental results of the microbial community sequencing test showed that: compared with the blank group, the abundance of probiotics in the three strain groups T1-5, T1-9, and 5B-1 Lactobacillaceae and Rikenellaceae was significantly increased, including Prevotellaceae in the 5B-1 group. So, the conclusion are as follows: Strains T1-5, T1-9 and 5B-1can promote intestinal peristalsis, regulate the structure of intestinal microbiota, and alleviate constipation in mice.