Objective To explore the molecular mechanisms of moxibustion in improving osteoporosis in rats using high-throughput micro-RNA (miRNA) sequencing analysis. Methods A total of 18 female Sprague-Dawley (SD) rats were randomly divided into an operation group (12 rats), which was subjected to ovariectomy to induce osteoporosis, and a sham operation control (SO) group (6 rats). The 12 osteoporosis model rats were randomly divided into a model (OVX) group and a moxibustion (MOX) group (6 rats per group), which were treated with once-daily moxibustion at the “Shenshu” (BL23) and “Guanyuan” (CV4) acupoints for 20 minutes each time, for 12 weeks. Micro-computed tomography (CT) scans of the rat femur were taken to analyze the trabecular bone thickness (Tb.Th), trabecular separation (Tb.Sp), and bone volume total volume (BV/TV) ratio of the trabecular bone. Hematoxylin and eosin staining was used for morphological observation of tibial tissues. 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 to screen for differentially expressed miRNAs, which were subjected to functional enrichment analysis and target gene prediction. Results Micro-CT images showed that, compared with the OVX group, the MOX group had superior bone density, significant increases in the Tb.Th and BV/ TV score, and a significant decrease in Tb.Sp (P<0.05). ELISA indicated that, compared with the OVX group, the MOX group showed a significant decrease in serum ALP activity and a significant increase in serum OCN content (P<0.05). The miRNA sequencing result showed that 34 miRNAs were commonly expressed between the OVX group and the intervention group, and 15 miRNAs were commonly expressed between the OVX group and the intervention group. KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly enriched in signaling pathways involving MAPK, Ras, FoxO, TNF, and cancer-related microRNAs. For the top five most differentially expressed microRNAs, 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. Conclusions Acupuncture slowed the progression of osteoporosis, improved bone microstructure, and balanced bone metabolism in rats. This therapeutic effect may be achieved through regulation of the expression of microRNAs and their target genes; however, the underlying mechanism requires further exploration.