BACKGROUND: The traditional herbal medicine Atractylodes macrocephala Koidz. (A. macrocephala) is commonly utilized for alleviating symptoms associated with spleen deficiency, abdominal distension, diarrhea, and constipation. These pharmacological effects are attributed to a variety of active constituents. However, the specific bioactive compounds responsible for promoting defecation and gastrointestinal transit in A. macrocephala remain unidentified. METHODS: The primary polysaccharide characteristics of PAMK was elucidated by HPLC, FT-IR, and HGPGC. Efficacy of PAMK (0.07, 0.14, and 0.28 mg/g) on mice was evaluated in a spleen deficiency constipation mouse model by analyzing stool parameters, constipation-related physiological indexes, and SCFAs. The expression levels of 5-HTR, 5-HTR, and related receptor genes were examined by RT-qPCR, and neurotransmitters were examined using ELISA. Finally, the diversity of gut microbiota was analyzed with 16S rDNA sequencing. KEY RESULTS: The results showed that PAMK significantly reduced the gastrointestinal transport time and increased the number of fecal pellets and fecal water content in spleen deficiency constipation model mice. PAMK kept the balance of 5-HT, SCFAs, TPH-1, SERT, CgA, and neurotransmitter levels (VIP, SP, MTL) in mice colon. In addition, PAMK could regulate the abundance of gut microbiota such as Alistopes, Bacteroides, and Odoribacter in spleen deficiency constipation model mice gut. CONCLUSIONS AND INFERENCES: It can be concluded that PAMK effectively ameliorated the symptoms of spleen deficiency constipation in mice by modulating the expression of 5-HT and its associated receptors. The underlying mechanism was elucidated, providing a solid theoretical foundation for the therapeutic application of A. macrocephala in treating spleen deficiency constipation and offering potential for developing novel approaches to address this condition.