Currently, the use of stem cell transplantation for the treatment of osteoporosis is a popular research topic. In general, promotion of the osteogenic differentiation of stem cells is the goal of researchers. MicroRNAs (miRNAs of miRs) are critical regulatory factors of osteogenic induction, and miR-9 and miR-10 have been substantiated to be involved in the osteogenic process, yet the underlying mechanisms remain unclear. The present study investigated the role of miR-9 and miR-10 in the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and aimed to elucidate the mechanism of osteogenic differentiation. Using MTT assay and RT-PCR, we found that miR-9 expression was upregulated and miR-10 expression was downregulated during osteogenic differentiation. Moreover, as shown by the results of mineralization analysis and western blot analysis, the overexpression of miR-9 enhanced the osteogenic abilities and overexpression of miR-9 inhibited these abilities. Furthermore, miR-9 and miR-10 also affected the expression of Runt-related transcription factor 2 (Runx2) and extracellular signal-regulated kinase (ERK) pathway by western blot analysis. Based on these results, we conclude that miR-9 and miR-10 play key roles in regulating the differentiation of BMSCs.