Objective: Based on high-throughput sequencing technology for micro-RNA (miRNA), this study explores the molecular mechanisms of moxibustion in improving osteoporosis in rats. Methods: A total of 18 female SD rats were randomly divided into a sham operation group (6 rats) and an operation group (12 rats). The operation group was subjected to ovariectomy to induce osteoporosis (OP), and the 12 rats with successful modeling were randomly divided into a model group and an moxibustion group, with 6 rats in each group. The moxibustion group rats were treated with moxibustion at the “Shenshu” (BL23) and “Guanyuan” (CV4), once a day, for 20 minutes each time, for 12 weeks. Micro-CT scan the rat femur and image it, analyze the trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone volume fraction (BV/TV) of the trabecular bone, and HE staining was used to observe the morphology of tibia tissue. Serum alkaline phosphatase (ALP) and osteocalcin (OCN) levels were measured using ELISA. Three randomly selected rats from each group were used for miRNA high-throughput sequencing, and differentially expressed miRNAs were screened. The functional enrichment analysis and target gene prediction were performed for the differentially expressed miRNAs. Results: The Micro-CT images showed that the bone density of the moxibustion group was superior to that of the model group, and compared to the model group, the moxibustion group showed a significant increase in the thickness of the trabecular bone (Tb.Th), a significant rise in the bone volume/tissue volume (BV/TV) score, and a significant decrease in the trabecular separation (Tb.Sp) (P < 0.05). The ELISA results indicated that compared to the model group, the moxibustion group showed a significant decrease in serum ALP activity and a significant increase in serum OCN content (P < 0.05). The miRNA sequencing results showed that 34 miRNAs were commonly expressed between the model group and the intervention group, and 15 miRNAs were commonly expressed between the model group and the intervention group. The KEGG pathway enrichment analysis showed that the expression of different genes was mainly enriched in signaling pathways such as MAPK, Ras, FoxO, TNF, and cancer-related microRNAs. For the top 5 differentially expressed microRNAs with the highest significance, namely miR-153-5p, miR-201-5p, miR-449c-5p, miR-451-3p, and miR-153-3p, target gene predictions yielded 10 major targets: Ebf2, Rtn4, Fbxl3, Naa15, Vamp2, Daam1, Akap6, Camta1, Ptprz1, and Lamp1. Conclusion: Acupuncture can slow down the progression of OP, improve bone microstructure, and balance bone metabolism. This therapeutic effect may be achieved by regulating the expression of microRNAs and their target genes, and a more detailed mechanism awaits further exploration.