Bone marrow mesenchymal stem cells (BMMSCs) are pluripotent stem cells, and the osteogenic differentiation of BMMSC5 has been drawing attention for a long time. Bone formation is regulated by numerous molecular and cellular signaling pathways, and the differentiation of BMMSCs is controlled by a well-defined genetic program. In the present study, we isolated BMMSC5 from the bone cavities of wild-type (WT) and microRNA-21 knock-out (miR-21-KO) mice and found that miR-21 was significantly upregulated during the osteogenic differentiation of BMMSC5. Under osteoinductive conditions, ALP staining and alizarin red staining showed that the bone formation of BMMSCs from miR-21-KO mice was less than that of BMMSC5 from WT mice. Consistently, RT-PCR and western blotting revealed that ALP and Runx2 expression levels in miR-21-KO mice were downregulated compared with those in WT mice. Meanwhile, the calvarial bone defects of miR-21-KO mice showed less newly formed bone than did those of WT mice. Additionally, the Smad7-Smad1/5/8-Runx2 axis showed the same tendency; Smad7 over expression and the expression of phosphorylated Smad1/5/8 complex decreased when miR-21 was knocked down. We identified a novel mechanism by which microRNA-21 (miR-21) promotes the bone formation of BMMSCs and found that this process is regulated, in part, by the Smad7-Smad1/5/8-Runx2 pathway. (C) 2017 Elsevier Inc. All rights reserved.