Apoptosis induced by endoplasmic reticulum (ER) stress plays a crucial role in mediating brain damage after ischemic stroke. Recently, Hes1 (hairy and enhancer of split 1) has been implicated in the regulation of ER stress, but whether it plays a functional role after ischemic stroke and the underlying mechanism remain unclear. In this study, using a mouse model of ischemic stroke via transient middle cerebral artery occlusion (tMCAO), we found that Hes1 was induced following brain injury, and that siRNA-mediated knockdown of Hes1 increased the cerebral infarction and worsened the neurological outcome, suggesting that Hes1 knockdown exacerbates ischemic stroke. In addition, mechanistically, Hes1 knockdown promoted apoptosis and activated the PERK/eIF2α/ATF4/CHOP signaling pathway after tMCAO. These results suggest that Hes1 knockdown promotes ER stress-induced apoptosis. Furthermore, inhibition of PERK with the specific inhibitor GSK2606414 markedly attenuated the Hes1 knockdown-induced apoptosis and the increased cerebral infarction as well as the worsened neurological outcome following tMCAO, implying that the protection of Hes1 against ischemic stroke is associated with the amelioration of ER stress via modulating the PERK/eIF2α/ATF4/CHOP signaling pathway. Taken together, these results unveil the detrimental role of Hes1 knockdown after ischemic stroke and further relate it to the regulation of ER stress-induced apoptosis, thus highlighting the importance of targeting ER stress in the treatment of ischemic stroke.