Prevention of cranial radiation-induced morbidity following the treatment of primary and metastatic brain cancers, including long-term neurocognitive deficiencies, remains challenging. Previously, we have shown that inhibition of glycogen synthase kinase 3β (GSK3β) results in protection of hippocampal neurons from radiation (IR)-induced apoptosis and attenuation of neurocognitive dysfunction resulting from cranial IR. In this study, we examined whether regulation of the repair of IR-induced DNA damage is one of the mechanisms involved in the radioprotective effects of neurons by inhibition of GSK3β. Specifically, this study showed that inhibition of GSK3β accelerated double strand-break (DSB) repair efficiency in irradiated mouse hippocampal neurons, as assessed by the neutral comet assay. This coincided with attenuation of IR-induced γ-H2AX foci, a well characterized in situ marker of DSBs. To confirm the effect of GSK3 activity on the efficacy of DSB repair, we further demonstrated that biochemical or genetic inhibition of GSK3 activity resulted in enhanced capacity in nonhomologous end-joining-mediated repair of DSBs in hippocampal neurons. Importantly, none of these effects were observed in malignant glioma cells. Taken together, these results suggested that enhanced repair of IR-induced DNA damage may be a novel mechanism by which inhibition of GSK3β specifically protects hippocampal neurons from IR-induced apoptosis. Furthermore, these findings warrant future investigations of the molecular mechanisms underlying the role of GSK3β in the DSB repair of normal neurons and the potential clinical application of neuroprotection with GSK3β inhibitors during cranial IR. © The Author(s) 2011.