Abstract: Objective: The study was to investigate the effect of Lacticaseibacillus casei on gastrointestinal regulatory peptide levels and gut microbiota in constipated mice in order to provide a new basis for constipation treatment. Methods: The constipated mice were modeled by intragastric administration of diphenoxylate solution. And these mice were then respectively treated with low (1×10⁶ CFU/mL), medium (1×10⁸ CFU/mL) and high (1×10¹⁰ CFU/mL) doses of L. casei. Intestinal functions were measured. Gastrointestinal regulatory peptide levels and their relationship with intestinal flora, and differences of gut microbiota among groups were analyzed. The effect of constipation on mice and the relieving of L. casei on constipation were explored. Results: Compared with the normal group, mice in the model group showed a prolonged time to the first black stoo, decreased small intestinal propulsion rate, and extremely significantly lower serum levels of gastrointestinal regulatory peptides as AChE, ET-1, MTL, SP and VIP (P<0.01). Those parameters demonstrated varying degrees of recovery trend in the mice after L. casei treatment, additionally the recovery trend exhibited more significant (P<0.05) in the high dose group. Heatmap analysis revealed that SP and ET-1 levels were positively correlated with unclassified_f__Lachnospiraceae (P<0.05). SP content was positively related to Lachnospiraceae_NK4A136_group (P<0.05) and Alloprevotella (P<0.01), while SS level was negatively correlated with unclassified_f__Lachnospiraceae (P<0.05) and Lachnospiraceae_NK4A136_group (P<0.01). Analysis of species differences among groups suggested that L. casei could increase the abundances of Staphylococcus_lentus_g__Staphylococcus (P<0.01), uncultured_bacterium_g__Alistipes (P<0.05) and Bacteroides_caecimuris (P<0.001) in the intestinal flora of mice, whereas it reduced the abundances of uncultured_bacterium_g__Prevotellaceae-UCG-001 (P<0.001) and unculture_bacterium_g__Odoribacter (P<0.01). Conclusion: L. casei was able to alleviate constipation by regulating the gut microbiota, increasing the number of beneficial bacteria, promoting the secretion of digestive juices and the contraction of gastrointestinal smooth muscle, and accelerating gastrointestinal motility.