This study demonstrated that 1,25-OH D stimulated odontoblastic differentiation of hDPSCs in vitro in a dose-dependent manner. The high DSP + cell number and a mild increase in ALP activity suggest that DPSCs treated with 0.1 μM 1,25-OH D are in the later stage of odontoblastic differentiation. The results confirm that 1,25-OH D-added cocktail medium provides a sufficient microenvironment for the odontoblastic differentiation of hDPSCs in vitro.