Whole brain irradiation (WBI) has become an indispensible tool in the treatment of head and neck cancer, and it has greatly improved patient survival rate and total survival time. In addition, prophylactic cranial irradiation (PCI) has dramatically decreased the incidence of brain metastatic carcinoma. However, WBI may induce temporary functional deficits or even progressive, irreversible cognitive dysfunction that compromises the quality of life for survivors. Unfortunately, the exact molecular mechanisms for cognitive damage remain elusive, and no treatment or preventative measures are available for use in the clinic. In the present study, the nuclear factor of activated T cells isoform 4 (NFAT3/c4) was found to play a vital role in excitotoxic hippocampus cell apoptosis induced by radiation. Sprague-Dawley (SD) rats received 20 Gy WBI, after which we detected NFAT3/c4-mediated excitotoxicity. We found that radiation caused hippocampus excitotoxicity, resulting from overactivation of the N-methyl-D-aspartate receptor (NMDAR) and always accompanied by subsequent elevation of the intracellular calcium level and activation of calcineurin (CaN). P-NFAT3/c4 was the principal downstream target of CaN, including regulation of its nuclear translocation as well as transcriptional activities. Radiation recruited NMDAR/NFAT3/c4 activation and subsequent Bax induction in hippocampus cells. Once treated with the NFAT3/c4 inhibitor 11R-VIVIT peptide pre-irradiation, hippocampal proliferation and neuron survival (dentate gyrus cells in particular) were protected from radiation-induced injury, resulting in inhibition of the apoptosis marker Bax. Our principal aim was to illuminate the role of NFAT3/c4-mediated excitotoxicity in hippocampal apoptosis during radiation-induced brain injury. This study is the first time that radiation-induced activation of NFAT3/c4 has been recorded, and our results suggest that NFAT3/c4 may be a novel target for prevention and treatment of radiation-induced brain injury.