To explore the relieving effect of compound Lactobacillus (Lactobacillus rhamnosus LrGG-100, Bifidobacterium longum BL-11, Lactobacillus acidophilus LA-99 and Lactobacillus casei LC-88) for slow transit constipation in mice, loperamide hydrochloride was used to establish a mouse model of constipation. Male BALB/c mice were randomly divided into the healthy group, constipation group, positive group, and low- and high-dose compound lactobacillus groups. After 21 days of gavage with corresponding contents, the mice were sacrificed for sampling, and their gastrointestinal function indicators such as the small intestine propulsion rate, levels of secreted serum cytokines, contents of gastrointestinal regulatory peptides, and transcription levels of AQP3 and c-kit were measured. The results showed that compared with the constipation group, the low- and high-dose compound lactobacillus groups had higher small intestine propulsion rate (increased by 61.45%, p<0.05; 1.22 times, p<0.05), shortened defecation time (decreased by 21.42%, p<0.05; 29.14%, p<0.05), increased number of defecation (increased by 1.5 times p<0.05; 1.52 times, p<0.05), and increased weight of stool (increased by 1.13 times, p<0.05; 1.25 times, p<0.05) in, the transcription levels of AQP3 and c-kit genes of the high-dose group were 1.82 times (p<0.05) and 2.00 times (p<0.05), respectively, those of the constipation group. Intragastric administration of compound Lactobacillus could increase the contents of substance P and motilin in the serum, and decrease the contents of vasoactive intestinal peptides, somatostatin and endothelin-1. Compound Lactobacillus could alleviate constipation symptoms. This effect may be related to the level of gastrointestinal regulatory peptides and the expressions of AQP3 and c-kit.