Endoplasmic reticulum (ER) stress plays a critical role in mediating ischemia/reperfusion (I/R) damage in the brain. Our previous study showed that Chrysophanol (CHR) alleviated cerebral ischemic injury in mice and nuclear factor-κB (NF-κB) involved in its neuroprotective effect, but the precise mechanism remains not fully understood. The present study investigated the effect of CHR treatment on I/R-induced ER stress. Mice were subjected to middle cerebral artery occlusion (MCAO) for 45 min and received either vehicle or CHR (0.1 mg/kg) for 14 days after reperfusion. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was used to detect apoptotic cells in penumbral tissue. The expression of ER stress-related factors including glucose-regulated protein 78 (GRP78), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), CCAAT-enhancer-binding protein homologous protein (CHOP), and caspase-12 as well as inhibitory κB-α (IκB-α), the inhibitor of NF-κB, was assessed. Our results demonstrated that CHR treatment reduced MCAO-induced upregulation of GRP78, p-eIF2α, CHOP, and caspase-12 in the ischemic brain. Moreover, the TUNEL-positive neuronal cells, which were colocalized with CHOP and caspase-12, decreased in response to CHR treatment, indicating that CHR protects against I/R injury by inhibiting ER stress-associated neuronal apoptosis. In addition, CHR reversed the decrease in IκB-α level induced by MCAO, which was attributed at least in part to the attenuation of translational inhibition induced by eIF2α phosphorylation, indicating that CHR exerts anti-inflammatory effects following I/R by inhibiting ER stress response. These results suggest that attenuation of ER stress may be involved in the mechanisms of neuroprotective effects of CHR.