Abstract Purpose: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2α (eIF-2α). There have been no reports to date about the role of PKR in radiation sensitivity. Experimental Design: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-κB (NF-κB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. Results: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR−/− MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-κB and Akt because both NF-κB and Akt are activated after ionizing radiation in PKR+/+ but not PKR−/− cells. Conclusions: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-κB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.