Background and Purpose RACK1 (receptor for activated protein kinase C 1) is an integral component of ribosomes with neuroprotective functions. The goal of this study was to determine the role of RACK1 in cerebral ischemia-reperfusion (I/R) injury and the underlying mechanisms. Methods A middle cerebral artery occlusion/reperfusion model in adult male Sprague Dawley rats (250-280 g) was established, and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation to mimic I/R injury in vitro. Expression vectors encoding wild-type RACK1 and RACK1 with T50A mutation (T50A) were constructed and administered to rats by intracerebroventricular injection. Results The potential role of RACK1 in cerebral I/R injury was confirmed by the decreased protein levels of RACK1 within penumbra tissue, especially of neurons. Second, there was an increase in the phosphorylation ratio of RACK1 at the threonine/serine residues at 1.5 hours after middle cerebral artery occlusion onset. Third, based on site-specific mutagenesis, we identified T50 as a key site for RACK1 phosphorylation during I/R. Fourth, wild-type RACK1 overexpression reduced infarct size, neuronal death, neuronal tissue loss, and neurobehavioral dysfunction, while RACK1 (T50A) overexpression exerted opposite effects. Finally, we found that RACK1 phosphorylation at T50 induced a loss of ribosomal RACK1, which switched RACK1 from beclin-1 translation inhibition to autophagy induction following I/R. Conclusions RACK1 phosphorylation may be a potential intervention target for neurons during I/R; thus, exogenous supplementation of RACK1 may be a novel approach for ameliorating I/R injury.