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Background Runt-related transcription factor 2 (RUNX2) plays a key role in the osteogenic differentiation. The study aimed to explore the effect of miR-153-3p on the osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) and confirm its targeting effect on RUNX2. Results During the osteogenic differentiation of hBMSCs, the expression of miR-153-3p decreased at 1 d and reached a low peak at 3 d. After miR-153-3p mimic or inhibitor transfection, miRNA-153-3p expression level of hBMSCs dramatically increased or decreased, but the cell viability and proliferation ability were not notably changed. After miR-153-3p inhibitor transfection, the osteogenic differentiation was notably promoted which was confirmed with Alizarin red staining, alkaline phosphatase (ALP) activity and Western blot for RUNX2 and Collagen I, while miR-153-3p mimic transfection, the osteogenic differentiation was notably suppressed. The result of luciferase reporter gene experiment verified that RUNX2 was the target gene of miR-153-3p. Conclusion The results of this study indicate that the expression of miR-153-3p regulates the osteogenic differentiation of hBMSCs by targeting RUNX2.