In the bone morphogenetic protein (BMP) family, BMP9 is the strongest inducer of osteogenic differentiation in mesenchymal stem cells. Recent studies have suggested that the miR-30 family regulates cell proliferation and osteoblastic differentiation. In the present study, we found that expression of only one miR-30 family member, miR-30a, first decreased and then increased during BMP9-induced osteogenic differentiation. Cell proliferation assays revealed that miR-30a had no effect on the proliferation of C3H10T1/2 cells. However, over-expression of miR-30a led to expression of an early osteogenic marker and a reduction in Runx2 expression. In addition, we observed decreases in the expression of late osteogenic markers and osteopontin, as well as calcium deposition. Dual-luciferase reporter assays indicated that this process might be mediated by suppressing Runx2 protein expression. In vivo stem cell implantation revealed inhibition of BMP9-induced ectopic bone formation and matrix mineralization by miR-30a. This study provides a better understanding of the molecular mechanisms through which miR-30a negatively regulates BMP9-induced osteogenic differentiation.