Many researches on tissue stem cells have been developed in the field of regenerative medicine. Some indicated that cultured dental pulp cells secreted dentin matrix. We transplanted subcultured human dental pulp cells, using alginate as a scaffold, into the subcutaneous layer of nude mice. By adding β-glycerophosphate into the culture media, we observed the expression of dentin sialophosphoprotein mRNA coding dentinsialoprotein (DSP). The increase of alkaline phosphatase, which is an indicative marker for odontoblast differentiation, was demonstrated. After 6 weeks of transplantation, formation of certain radiopaque structures was observed in situ subcutaneously. The immunohistochemical and fine structure study identified the expression of type I collagen, type III collagen and DSP in the calcifying tissue. Also, isolated odontoblast-like cells relating to dentin-like hard tissue formation and scattering autolyzing apoptotic cells were found. The study elucidates that subcultured dental pulp cells actively differentiate secretory cells and induce calcification in the alginate scaffold.