OBJECTIVE: To investigate the regulatory effect of moxibustion, a traditional Chinese medicine therapy, on bone metabolism in ovariectomized (estrogen-deficient) mice and to explore its underlying mechanisms. METHODS: Female C57BL/6J mice of 12 weeks old were randomly assigned to five groups, including a sham operation control group (SHAM), an ovariectomy group (OVX), a group given ovariectomy and broad-spectrum antibiotics (OVX-A), a group given ovariectomy and moxibustion (OVX-M), and a group given ovariectomy, broad-spectrum antibiotics, and moxibustion (OVX-A-M), with 5 mice in each group. Then, 4 weeks post-surgery, the mice in each group received broad-spectrum antibiotics and/or moxibustion intervention for an additional 4 weeks. After that, the mice were sacrificed, and samples were collected. Micro-CT was used to assess bone volume parameters in the distal femurs, including bone volume/tissue fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness (Ct.Th). Targeted metabolomics was used to measure serum tryptophan metabolites, and qPCR was performed to quantify serotonin (SER) receptors RESULTS: Compared with the SHAM mice, the OVX mice exhibited significant deterioration in bone microarchitecture, showing decreased BV/TV ([10.57 ± 2.82]% vs. [4.20 ± 0.96]%, CONCLUSION: Moxibustion therapy can inhibit postmenopausal bone loss, potentially by regulating gut microbiota-derived SER, activating the 5-HT2A receptor, and promoting the osteogenic differentiation of BMSCs.