Core binding factor beta (Cbf beta) is a non-DNA binding cofactor of Runx2 that potentiates DNA binding. Previously, it has been reported that Cbf beta plays an essential role in osteogenic differentiation and skeletal development by inhibition adipogenesis. Here, we delivered the recombinant Cbf beta protein into human mesenchymal stem cells (MSCs) and triggered osteogenic lineage commitment. The efficient delivery of Cbf beta was achieved by fusing 30Kc19 protein, which is a cell-penetrating protein derived from the silkworm. After the production of the recombinant Cbf beta-30Kc19 protein in theEscherichia coliexpression system, and confirmation of its intracellular delivery, MSCs were treated with the Cbf beta-30Kc19 once or twice up to 300 mu g/ml. By investigating the upregulation of osteoblast-specific genes and phenotypical changes, such as calcium mineralization, we demonstrated that Cbf beta-30Kc19 efficiently induced osteogenic differentiation in MSCs. At the same time, Cbf beta-30Kc19 suppressed adipocyte formation and downregulated the expression of adipocyte-specific genes. Our results demonstrate that the intracellularly delivered Cbf beta-30Kc19 enhances osteogenesis in MSCs, whereas it suppresses adipogenesis by altering the transcriptional regulatory network involved in osteoblast-adipocyte lineage commitment. Cbf beta-30Kc19 holds great potential for the treatment of bone-related diseases, such as osteoporosis, by allowing transcriptional regulation in MSCs, and overcoming the limitations of current therapies.