Identification of medical countermeasures (MCM) to mitigate radiation damage and/or protect first responders is a compelling unmet medical need. The prostaglandin E₂ (PGE₂) analog, 16,16 dimethyl-PGE₂ (dmPGE₂), has shown efficacy as a radioprotectant and radiomitigator that can enhance hematopoiesis and ameliorate intestinal mucosal cell damage. In this study, we optimized the time of administration of dmPGE₂ for protection and mitigation against mortality from the hematopoietic acute radiation syndrome (H-ARS) in young adult mice, evaluated its activity in pediatric and geriatric populations, and investigated potential mechanisms of action. Windows of 30-day survival efficacy for single administration of dmPGE₂ were defined as within 3 h prior to and 6–30 h after total-body γ irradiation (TBI). Radioprotective and radio-mitigating efficacy was also observed in 2-year-old geriatric mice and 6-week-old pediatric mice. PGE₂ receptor agonist studies suggest that signaling through EP4 is primarily responsible for the radioprotective effects. DmPGE₂ administration prior to TBI attenuated the drop in red blood cells and platelets, accelerated recovery of all peripheral blood cell types, and resulted in higher hematopoietic and mesenchymal stem cells in survivor bone marrow. Multiplex analysis of bone marrow cytokines together with RNA sequencing of hematopoietic stem cells indicated a pro-hematopoiesis cytokine milieu induced by dmPGE₂, with IL-6 and G-CSF strongly implicated in dmPGE₂-mediated radioprotective activity. In summary, we have identified windows of administration for significant radio-mitigation and radioprotection by dmPGE₂ in H-ARS, demonstrated survival efficacy in special populations, and gained insight into radioprotective mechanisms, information useful towards development of dmPGE₂ as a MCM for first responders, military personnel, and civilians facing radiation threats.