Adult human dental pulp contains stem cells (DPSCs) that are capable of differentiation into osteoblasts, odontoblasts, adipocytes, and neuronal-like cells. Because these cells have potential use in tissue regeneration, herein we characterized the response of DPSC lines to ionizing radiation (IR). These DPSC lines have been developed from the extracted molars of healthy donors. DPSCs were cultivated in a unique media supplemented with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Since tissue homeostasis depends on a precise balance among cell proliferation, senescence, and cell death, we explored the effects of IR (2-20 Gy) on the proliferative activity of DPSCs and the molecular pathways involved. Even the highest dose used (20 Gy) did not induce DPSC apoptosis. After irradiation with doses of 6 and 20 Gy, DPSCs accumulated in the G2 phase of the cell cycle. DPSCs responded to IR (20 Gy) with senescence detected as SA-β-galactosidase positivity, beginning on the third day after irradiation. Twenty-four hours after irradiation, p53 and its serine 15 and 392 phosphorylated forms were detected. At this time, p21 (WAF1) was induced. Increases in protein p16 were observed from the third day following irradiation and continued till the end of the examination (Day 13). We conclude that DPSCs respond to IR-induced damage by permanent cell cycle arrest in the G2 phase and by stress-induced premature senescence.