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As a regulator of osteogenesis, microRNA-218 (miR-218) is reported to promote osteogenesis of mesenchymal stem cells (MSCs). However, the in vivo osteogenic effect of miR-218 remains elusive. In this study, miR-218 was confirmed to promote osteogenic differentiation of MSCs by stimulating the alkaline phosphatase activity, calcium nodule formation, and osteogenic marker gene expression. For in vivo study, the miR-218-overexpressing BMSCs were locally administrated into the fracture sites in a femur fracture mouse model. Based on the X-rays, micro-computed tomography, mechanical testing, histology, and immunohistochemistry examinations, miR-218 overexpression improved new bone formation and accelerated fracture healing. These findings suggest that miR-218 may be a promising therapeutic target for bone repair in future clinical applications.