The hypoxia condition was expected to be suitable for the establishment and maintenance of human dental pulp cells (hDPCs), because they reside in a low-oxygen environment in vivo. Therefore, we presently examined the effects of hypoxia on the proliferation and differentiation of hDPCs in vitro. hDPCs grown under 3% O(2) showed a significantly higher proliferation rate than those under 21% O(2). Then, we prepared hypoxic cultures of hDPCs from older patients' teeth having inflammation and succeeded in recovering and expanding a small number of hDPCs. These cells were confirmed to have capability for osteo/odontogenic differentiation. Hypoxia suppressed the osteo/odontogenic differentiation of hDPCs in vitro and increased the number of cells expressing STRO-1, an early mesenchymal stem cell marker. This simple method will increase the possibility to obtain living hDPCs from damaged and/or aged tissues, from which it is ordinarily difficult to isolate living stem cells with differentiation capability.