Mesenchymal stromal cells (MSCs) are radio-resistant bone marrow progenitors that support hematopoiesis and its re-constitution following total body irradiation. MSCs reside in hypoxic niches within the bone marrow and tumor microenvironments. The DNA Damage Response (DDR) represents a network of signaling pathways that enable cells to activate biological responses to DNA damaging agents. Hypoxia-mediated alterations in the DDR contribute to the increased radio-resistance of hypoxic cancer cells, limiting therapeutic efficacy. The DDR is important in mediating mouse MSC radio-resistance. However, the effects of hypoxia on MSC radio-resistance are currently unknown. In this report, hypoxia was found to (i) increase MSC proliferation rate and colony size; (ii) increase long-term survival post irradiation and (iii) improve MSC recovery from IR-induced cell cycle arrest. DNA DSB repair in MSCs was up-regulated in hypoxia, accelerating the resolution of highly genotoxic IR-induced DNA double-strand breaks (DSBs). In addition, HIF-1α was found to contribute to this enhanced DSB repair by regulating (i) the expression of DNA ligase IV and DNA-PKcs and (ii) Rad51 foci formation in response to DNA DSBs in hypoxic MSCs. We have demonstrated, for the first time, that hypoxia enhances mouse MSC radio-resistance in vitro. These findings have important implications for our understanding of MSC functions in supporting allogeneic bone marrow transplantation and in tumorigenesis. Stem Cells 2014