Our previous reports indicated that (+)-cholesten-3-one induces osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs) by activating vitamin D receptor (VDR). However, whether and how miRNAs modulate osteogenic differentiation induced by (+)-cholesten-3-one have not been explored. In this study, miRNA array profiling and further validation by quantitative real-time PCR revealed that miR-351 was downregulated during (+)-cholesten-3-one-induced osteogenic differentiation of MSCs. Overexpression of miR-351 by miR-351 precursor transfection markedly inhibited the expression of osteoblast-specific genes, such as alkaline phosphatase (ALP), collagen type II, osteopontin (OPN), and runt-related transcription factor 2 (RUNX2), which consequently decreased a number of calcium mineralized nodules. Inhibition of miR-351 function by anti-miR-351 promoted expression of osteoblast-specific genes. Our results suggest that miR-351 is a negative regulator of osteoblast differentiation of MSCs induced by (+)-cholesten-3-one. Target prediction analysis tools and experimental validation by luciferase 3'UTR reporter assay identified VDR as a direct target of miR-351. miR-351 inhibited the expression of the VDR, which played a critical role in the control of osteogenic differentiation of MSCs. Importantly, overexpression of VDR significantly abolished the inhibitory effect of miR-351 on (+)-cholesten-3-one induced osteogenic differentiation. Taken together, our results demonstrate that miR-351 negatively regulates osteoblast differentiation of MSCs induced by (+)-cholesten-3-one through targeting VDR. These findings provid evidence that miR-351 can bea possible therapeutic target for bone repair and regeneration.