Mesenchymal stromal cells (MSCs) are radioresistant bone marrow progenitors that support hematopoiesis and its reconstitution 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 radioresistance of hypoxic cancer cells, limiting therapeutic efficacy. The DDR is important in mediating mouse MSC radioresistance. However, the effects of hypoxia on MSC radioresistance are currently unknown. In this report, hypoxia was found to (a) increase MSC proliferation rate and colony size; (b) increase long-term survival post-irradiation (IR), and (c) improve MSC recovery from IR-induced cell cycle arrest. DNA double-strand break (DSB) repair in MSCs was upregulated in hypoxia, accelerating the resolution of highly genotoxic IR-induced DNA DSBs. In addition, HIF-1 alpha was found to contribute to this enhanced DSB repair by regulating (a) the expression of DNA ligase IV and DNA-PKcs and (b) Rad51 foci formation in response to DNA DSBs in hypoxic MSCs. We have demonstrated, for the first time, that hypoxia enhances mouse MSC radioresistance in vitro. These findings have important implications for our understanding of MSC functions in supporting allogeneic bone marrow transplantation and in tumorigenesis.