Inhibition of protein synthesis executed at the initiation step is the overall response of cells during stress. Here, we evaluated the effect of gamma radiation induced oxidative stress on protein synthesis in human K562 cells. In erythroid cells such as K562, the heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase, also called the heme-regulated inhibitor (HRI), is abundant and is instrumental in regulating protein synthesis. We, therefore, examined the modulation of expression and activity of HRI in K562 cells at various time points following their exposure to 6 Gy of gamma radiation. Radiation-induced oxidative stress was reflected by a dose-dependent increase in the intracellular reactive oxygen species with time up to 6 h. Further, cell membrane damage in terms of lipid peroxidation and lipid hydroperoxide formation was also observed. Interestingly, radiation induced oxidative stress led to a significant decrease in the rate of protein synthesis caused due to induced activation as well as expression of HRI within 1 h. Furthermore, radiation exposure also caused increased expression of heat shock protein 90 (Hsp90) in 1 h. These results have demonstrated shutdown of global protein synthesis in K562 cells during radiation induced oxidative stress, mediated by overexpression and activation of HRI possibly caused by Hsp90.