The role of cytosolic calcium oscillation has long been recognized in the regulation of cellular and molecular interactions. Information embedded in calcium oscillation can provide molecular cues for cell behavior such as cell differentiation. Although calcium dynamics are versatile and likely to depend on the cell type, the calcium dynamics in human mesenchymal stem cells (hMSCs) and its role in differentiation are yet to be fully elucidated. In the present study we characterized the calcium oscillation profiles in hMSCs before and after subjecting the cells to the osteoinductive factors. Our findings indicate that the calcium spikes decreased rapidly with osteodifferentiation to a level observed in terminally differentiated human osteoblasts. In addition, the calcium oscillations appear to serve as a bidirectional signal during hMSC differentiation. While an altered calcium oscillation pattern may be an indicator for hMSC differentiation, it is also likely to be involved in directing hMSC differentiation. Treatment of hMSCs with a noninvasive electrical stimulation, for example, not only altered the calcium oscillations but also facilitated osteodifferentiation. Regulation of calcium oscillation by external physical stimulation could amplify hMSC differentiation into a tissue-specific lineage and may offer an alternate biotechnology to harness the unique properties of stem cells.--Sun, S., Liu, Y., Lipsky, S., Cho, M. Physical manipulation of calcium oscillations facilitates osteodifferentiation of human mesenchymal stem cells.