Exposure to high-dose ionizing radiation can lead to life-threatening injuries and mortality. Bone marrow is the most sensitive organ to radiation damage, resulting in the hematopoietic acute radiation syndrome (H-ARS) with the potential sequelae of infection, hemorrhage, anemia, and death if untreated. The development of medical countermeasures (MCMs) to protect or mitigate radiation injury is a medical necessity. In our well-established murine model of H-ARS we have demonstrated that the prostaglandin E₂ (PGE₂) analog 16,16 dimethyl-PGE₂ (dmPGE₂) has survival efficacy as both a radioprotectant and radiomitigator. The purpose of this study was to investigate the pharmacokinetics (PK) and biodistribution of dmPGE₂ when used as a radioprotector in irradiated and non-irradiated inbred C57BL/6J mice, PK in irradiated and non-irradiated Jackson Diversity Outbred (JDO) mice, and the PK profile of dmPGE₂ in non-irradiated non-human primates (NHPs). The C57BL/6J and JDO mice each received a single subcutaneous (SC) dose of 35 ug of dmPGE₂ and were randomized to either receive radiation 30 min later or remain non-irradiated. Plasma and tissue PK profiles were established. The NHP were dosed with 0.1 mg/kg by SC administration and the PK profile in plasma was established. The concentration time profiles were analyzed by standard non-compartmental analysis and the metrics of AUC_0–Inf, AUC_60–480 (AUC from 60–480 min), C_max, and t_1/2 were evaluated. AUC₆₀–₄₈₀ represents the postirradiation time frame and was used to assess radiation effect. Overall, AUC_0–Inf, C_max, and t_1/2 were numerically similar between strains (C57BL/6J and JDO) when combined, regardless of exposure status (AUC_0–Inf: 112.50 ng·h/ml and 114.48 ng·h/ml, C_max: 44.53 ng/ ml and 63.96 ng/ml; t_1/2: 1.8 h and 1.1 h, respectively). PK metrics were numerically lower in irradiated C57BL/6J mice than in non-irradiated mice [irradiation ratio: irradiated values/non-irradiated values = 0.71 for AUC_60–480 (i.e., 29% lower), and 0.6 for t_1/2]. In JDO mice, the radiation ratio was 0.53 for AUC_60–480 (i.e., 47% lower), and 1.7 h for t_1/2. The AUC_0–Inf, C_max, and t_1/2 of the NHPs were 29.20 ng·h/ml, 7.68 ng/ml, and 3.26 h, respectively. Despite the numerical differences seen between irradiated and non-irradiated groups in PK parameters, the effect of radiation on PK can be considered minimal based on current data. The biodistribution in C57BL/6J mice showed that dmPGE₂ per gram of tissue was highest in the lungs, regardless of exposure status. The radiation ratio for the different tissue AUC_60–480 in C57BL/6J mice ranged between 0.5–1.1 (50% lower to 10% higher). Spleen, liver and bone marrow showed close to twice lower exposures after irradiation, whereas heart had a 10% higher exposure. Based on the clearance values from mice and NHP, the estimated allometric scaling coefficient was 0.81 (95% CI: 0.75, 0.86). While slightly higher than the current literature estimates of 0.75, this scaling coefficient can be considered a reasonable estimate and can be used to scale dmPGE₂ dosing from animals to humans for future trials.