Abstract Background and Purpose Previous studies have shown that TIGAR (TP53-induced glycolysis and apoptosis regulator) protects against cerebral ischemia/reperfusion injury via the improvement of the redox and energy homeostasis of neurons. TIGAR is found in the endoplasmic reticulum (ER) and nucleus but its role in ER stress is unclear. This study aimed to investigate the ER and nuclear translocation of TIGAR during ER stress and the influences of the nuclear TIGAR in cerebral ischemia/reperfusion-induced ER stress. Experimental Approach Mice were subjected to the middle cerebral artery occlusion/reperfusion. Cultured neurons were treated with oxygen and glucose deprivation, tunicamycin or thapsigargin. Key Results The increases in ATF4 target genes and ER stress-induced neuronal apoptosis were reduced by overexpression of TIGAR. Furthermore, increases in the localization of TIGAR and ATF4 to the nucleus were observed after in vitro and in vivo cerebral ischemia/reperfusion or ER stress models. The nuclear TIGAR interacts with ATF4 and inhibits the transcription of downstream pro-apoptotic genes of ATF4, resulting in protection against cerebral ischemia/reperfusion injury. Intriguingly, the translocation of TIGAR to the ER and nucleus and inhibition of the transcription of ATF4 is depend on Q141/K145 of TIGAR instead of its phosphatase activity and mitochondrial localization domains. Conclusion and Implications These results suggest that TIGAR translocates to the ER and the nucleus to interact with ATF4 after cerebral ischemia-reperfusion induced ER stress via its Q141/K145. The study uncovered a novel neuroprotective mechanism of TIGAR through regulating ER stress via a ATF4-mediated signaling pathway.