Background: 1,25-Dihydroxyvitamin D-3 (1,25-OH D-3) plays an important role in mineralized tissue metabolism, including teeth. However, few studies have addressed its role in odontoblastic differentiation of human dental pulp-stem cells (hDPSCs). Aim: This study aimed to understand the influence of various concentrations of 1,25-OH D-3 on the proliferation capacity and early dentinogenesis responses of hDPSCs. Materials and Methods: hDPSCs were obtained from the impacted third molar teeth. Monolayer cultured cells were incubated with a differentiation medium containing different concentrations of 1,25-OH D-3 (0.001, 0.01, and 0.1 mu M). All groups were evaluated by S-phase rate [immunohistochemical (IHC) bromodeoxyuridine (BrdU) staining], STRO-1 and dentin sialoprotein (DSP)+ levels (IHC), and alkaline phosphatase (ALP, enzyme-linked immunosorbent assay (ELISA)) levels. Results: The number of cells that entered the S-phase was determined to be the highest and lowest in the control and 0.001 mu M 1,25-OH D-3 groups, respectively. The 0.1 mu M vitamin D-3 group had the highest increase in DSP+ levels. The highest Stro-1 levels were detected in the control and 0.1 mu M 1,25-OH D-3 groups, respectively. The 0.1 mu M 1,25-OH D-3 induced a mild increase in ALP activity. Conclusions: This study demonstrated that 1,25-OH D-3 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 M 1,25-OH D-3 are in the later stage of odontoblastic differentiation. The results confirm that 1,25-OH D-3-added cocktail medium provides a sufficient microenvironment for the odontoblastic differentiation of hDPSCs in vitro.