Abstract: Radiation is an important therapeutic tool in the treatment of cancer. The tremendous development in radiotherapeutic techniques and dosimetry has made it possible to augment the patient survival. Therefore, attention has focused on long-range treatment side effects especially in relation to the neurocognitive changes. As cognitive health of an organism is considered to be maintained by the capacity of hippocampal neurogenesis, this study designed to evaluate the delayed effect of cranial irradiation on hippocampal neurogenesis, possible implication of oxidative stress and prophylactic action of melatonin in mice. One month after cranial irradiation (6 Gy, X-ray), changes in the population of immature and proliferating neurons in dentate gyrus were localized through the expression of the microtubule binding protein doublecortin (Dcx) and proliferation marker Ki-67. We found a substantial reduction in the Dcx and Ki-67 positive cells after irradiation. Melatonin pretreatment significantly ameliorated the radiation-induced decline in the Dcx and Ki-67 positive cells. In addition, profound increase in the 4-hydroxynonenal (4-HNE) and 8-hydroxy-2′-deoxyguanosine positive cells were reported in subventricular zone, granular cell layer and hilus after day 30 postirradiation. Immunoreactivity of these oxidative stress markers were significantly inhibited by melatonin pretreatment. To confirm the magnitude of free-radical scavenging potential of melatonin, we measured the in-vitro OH radical scavenging power of melatonin by electron spin resonance. Interestingly, the melatonin was capable of scavenging the OH radicals at very low concentration (IC50 = 214.46 nm). The findings indicate the possible benefit of melatonin treatment to combat the delayed side effects of cranial radiotherapy.