Abstract: The effects of five strains of Lactobacillus paracasei from Tibet on constipation and gut microbiota of mice were studied. One hundred and sixty 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 subgroup 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 h (P<0.05). The wet weight of feces in the T1-9 and 6108 groups increased significantly (P<0.01, 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-1 can promote intestinal peristalsis, regulate the structure of intestinal microbiota, and alleviate constipation in mice.