Rutin, a flavonoid glycoside, has been reported to exert neuroprotective effects. Loss of endogenous estrogen and dysregulation of the estrogen receptor (ER) signaling pathway are associated with the increased risk of stroke in women after menopause. This study was performed to investigate whether rutin could protect against cerebral ischemia by modulating the ER pathway. Ovariectomized (OVX) rats were given intraperitoneal injections of vehicle (dimethyl sulfoxide), rutin (100 mg/kg body mass) or 17β-estradiol (100 μg/kg body mass) for 5 consecutive days. Then, the rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h followed by a 24 h reperfusion to establish the cerebral ischemia–reperfusion (I/R) injury. We found that rutin improved the sensorimotor performance and recognition memory of rats subjected to I/R, decreased the infarct size, and attenuated neuron loss. Rutin treatment also increased the levels of ERα, ERβ, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), tropomyosin receptor kinase A (TrkA), TrkB, and phospho-cAMP-responsive element binding protein (p-CREB) in rat hippocampus and cerebral cortex. The protective effects of rutin were comparable to that of 17β-estradiol, and were partially blocked by ICI182780, an ER antagonist. The above results suggest that rutin preconditioning ameliorates cerebral I/R injury in OVX rats through ER-mediated BDNF–TrkB and NGF–TrkA signaling.