Abstract Pregnant adult C57BL/6J mice were randomly assigned to four groups: one sham and three irradiated groups that were exposed to β-irradiation from tritiated water (HTO) by a single intraperitoneal injection on Day 12.5 of gestation. The offspring received cumulative doses of 0.036, 0.071, and 0.213 Gy, respectively. The litters were observed for postnatal growth (body weight, brain weight), the development of four physiologic milestones (pinna detachment, eye opening, testes decent, vaginal opening), the acquisition age of several reflexes (cliff avoidance, air righting) and sensory functions (auditory startle, thermal reflex), movement and coordination functions and activity (pivoting, foot splay, continuous corridor activity), learning and memory performance (shock avoidance, conditioning reflex), and the density of CA1–CA4 hippocampal pyramidal neurons. Modest but significant dose-dependent neuronal death and functional impairment were seen in both 0.071 and 0.213 Gy groups. In conclusion, even prenatal low-dose β-radiation may impair murine central nervous system (CNS) development suggesting the potential importance of minimizing environmental exposure during human pregnancy.