Abstract:[Objective] The study aims to investigate the alleviating effect of Lactobacillus plantarum (L. plantarum) CCFM8661 on constipation in mice. [Method] One hundred mice were allocated into five groups (control, model, and low-, medium-, and high-dose groups) and administrated with corresponding agents by gavage for 14 d. On the last day, the mouse model of constipation was established by gavage of loperamide hydrochloride. The degree of constipation alleviation by L. plantarum CCFM8661 was evaluated through the determination of the defecation situation, short-chain fatty acids (SCFAs), and intestinal flora of mice. [Result] L. plantarum CCFM8661 effectively alleviated the symptoms of constipation in a dose-dependent manner. [Conclusion] L. plantarum CCFM8661 may relieve constipation via multi-level regulation. On the one hand, it improves the intestinal function by regulating the levels of neurotransmitters and the expression of inflammatory factors; on the other hand, it inhibits the growth of pathogenic bacteria by increasing the molar mass concentrations of SCFAs (acetic acid, propionic acid, and butyric acid) and decreasing the intestinal pH while reshaping the intestinal flora balance. The analysis of KEGG pathways further reveals that these effects may be closely related to biological processes such as glucose metabolism, amino acid metabolism, as well as the synthesis, transport, and cycling of a variety of substances, which synergistically exert their multi-pathway regulatory effects to alleviate constipation. This study further demonstrates the potential of L. plantarum CCFM8661 in alleviating constipation and provides a reference for the application of this strain.