It is well known that the interactions between epithelial and mesenchymal components are very important event for tooth development, and their coordination is crucial to tooth development. Recently it has been reported that human PDLSC might be a mesenchymal component for regeneration of periodontal tissue. In contrast, characteristics of human HERS cells and their roles in development and regeneration of tooth are not well elucidated. In this study, we investigated the phenotypes and the differentiation potentials of human HERS cells and PDLSCs. First of all, we simultaneously isolated human HERS cells and PDLSCs from the same periodontal ligament tissues. Hence we established the method for primary isolation of human HERS cells and PDLSC at the same time. Human HERS cells and PDLSCs showed different cell morphology and growth patterns from each other. In particular, human HERS cells showed epithelial cell-like morphology, while human PDLSCs showed fibroblast-like morphology. In the immunophenotype analysis of cell surface markers, human PDLSCs were positive for the mesenchymal stem cell markers but human HERS cells showed different expressions. Human PDLSCs were able to differentiate into osteoblasts and adipocytes by appropriate induction. On the other hand, human HERS cells acquired mesenchymal phenotypes by TGF-β1 through EMT. Taken together, our data suggest that human HERS cells and PDLSCs might play a role as a epithelial component or a mesenchymal component for the tooth regeneration,v respectively.