Aim Epigenetic features such as DNA methylation are increasingly being recognized as an important factor in the occurrence of many complex diseases. However, there are limited data on the DNA methylation changes in ischemic stroke patients. This study is designed to determine whether the DNA methylation status of estrogen receptor-α （ER-α） gene promoter is related to ischemic stroke. Methods 83 ischemic stroke patients and 94 control subjects were selected for research. The infarct size was recorded and the severity of neurological impairment was assessed by National Institute of Health Stroke Scale （NIHSS） and Barthel Index. For patients with carotid artery color Doppler ultrasound and brain magnetic resonance angiography （MRA）, Crouse score and plaque index were calculated to evaluate the severity of intracranial atherosclerosis. Morning fasting venous blood sample was taken for DNA extraction. The methylation status of ER-α gene promoter was measured by methylation specific polymerase chain reaction （MSP）. Results The ER-α gene promoter methylation frequency was higher in ischemic stroke group than in control group （42.2% vs 19.1%, P＜0.05）. Carotid artery color ultrasound examination was performed in 52 patients, and there were statistical differences in carotid intima-media thickness （CIMT）, Crouse score and plaque index among full-methylation, part-methylation and non-methylation subgroups （P＜0.05）. Brain MRA was performed in 57 patients, and the methylation frequency had tendency to increase with the severity of intracranial atherosclerosis （respectively 40.9%, 42.9%, 52.4%, 57.1%）, but there was no statistical significance （P>0.05）. According to the size of infarct, the patients was divided into small infarction group, middle infarction group and large infarction group, and methylation frequency increased in turn （respectively 32.8%, 56.3%, 77.8%）, the difference was statistically significant （P＜0.05）. There were statistical differences in NIHSS score and Barthel index among full-methylation, part-methylation and non-methylation subgroups （P＜0.05）. Conclusions The ER-α gene promoter methylation frequency is higher in ischemic stroke patients than in control group. The ER-α gene promoter methylation status is related to the severity of carotid atherosclerosis, infarct size and the severity of neurological impairment.