MicroRNAs (miRNAs) are novel key regulators of cellular differentiation. miR‑124 has been reported to regulate osteogenic differentiation of bone marrow‑derived mesenchymal stem cells (BMSCs). However, the specific mechanisms involved have not yet been fully elucidated. The present study aimed to investigate the effect of miR‑124 on osteogenic differentiation of BMSCs and its underlying mechanisms. In the present study, it was found that alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and the protein levels of osterix (Sp7) and runt‑related transcription factor 2 (Runx2) were significantly increased, whereas the expression of miR‑124 was decreased in a time‑dependent manner during osteogenic differentiation of BMSCs. Following overexpression of miR‑124 via transfection of miR‑124 mimics in BMSCs, Runx2 protein expression and ALP activity were significantly decreased. By contrast, inhibition of miR‑124 expression led to an increase in ALP activity and Runx2 expression. Sp7 expression was suppressed in BMSCs transfected with miR‑124 mimics while increased when miR‑124 expression was inhibited, indicating that miR‑124 regulates the expression of Sp7. Moreover, a luciferase reporter assay further verified that Sp7 is the direct target of miR‑124. Finally, the effect of miR‑124 inhibitor on promoting the differentiation of BMSCs was abolished following treatment with a small interfering RNA targeting Sp7. Taken together, the present study demonstrates that miR‑124 inhibits the osteogenic differentiation of BMSCs by targeting Sp7.