Electromagnetic fields (EMFs) have emerged as an effective treatment for osteoporosis. However, the specific mechanism underlying their therapeutic efficacy remains controversial. Herein, we confirm the pro-osteogenic effects of 15 Hz and 0.4-1 mT low-frequency sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs). Subsequent miRNA sequencing reveal that miR-34b-5p is downregulated in both the 0.4 mT and 1 mT SEMFs-stimulated groups. To clarify the role of miR-34b-5p in osteogenesis, BMSCs are transfected separately with miR-34b-5p mimic and inhibitor. The results indicate that miR-34b-5p mimic transfection suppress osteogenic differentiation, whereas inhibition of miR-34b-5p promote osteogenic differentiation of BMSCs. In vivo assessments using microcomputed tomography, H&E staining, and Masson staining show that miR-34b-5p inhibitor injections alleviate bone mass loss and trabecular microstructure deterioration in ovariectomy (OVX) rats. Further validation demonstrates that miR-34b-5p exerts its effects by regulating STAC2 expression. Modulating the miR-34b-5p/STAC2 axis attenuate the pro-osteogenic effects of low-frequency SEMFs on BMSCs. These studies indicate that the pro-osteogenic effect of SEMFs is partly due to the regulation of the miR-34b-5p/STAC2 pathway, which provides a potential therapeutic candidate for osteoporosis.