Abstract: Objective: The objective of this study was to evaluate the efficacy of Bifidobacterium animalis subsp. lactis Bi66 in alleviating constipation in mice and exploring its mechanism, thereby providing a high-quality strain for native culture resources. Methods: Male Balb/c mice were randomly divided into the control group, the constipation model group, the Bi66 group, and the BB12 group. Probiotics were orally administered to the mice for 14 days followed by loperamide administration to induce constipation. Fecal indexes, intestinal propulsive rate, and serum gastrointestinal regulatory peptides of mice were measured, and gut microbiota was analyzed by 16S rRNA gene sequencing. Result: Bifidobacterium animalis subsp. lactis Bi66 significantly increased the number of feces particles, fecal wet weight, and fecal water content (P<0.05). It also significantly reduced the time of first black stool (P<0.05) and improved the intestinal propulsive rate in constipated mice. Moreover, Bi66 significantly improved motilin (P<0.05), gastrin (P<0.01), and decreased vasoactive intestinal peptide and endothelin levels (P<0.05). The analysis of gut microbiota revealed that the administration of Bifidobacterium animalis subsp. lactis Bi66 enhanced both the abundance and diversity of intestinal flora in constipated mice. Specifically, Bi66 exhibited a remarkable reduction in Firmicutes while promoting an increase in Bacteroidetes in constipated mice. At the genus level, the relative abundances of Alistipes, Roseburia, Ligilactobacillus, Blautia, unclassified-Muribaculaceae, and Alloprevotella showed significant increase (P<0.05, P<0.01). Conclusion: Bifidobacterium animalis subsp. lactis Bi66 alleviated constipation in mice by regulating gastrointestinal regulatory peptides and gut microbiota.