Background: Electroacupuncture (EA) therapy, as a combination of electric stimulation and traditional acupuncture technology, is currently an important mean for treatment of ischemic stroke and post-stroke rehabilitation in China. Because of its remarkable therapeutic effect, it has been widely used in hospitals and clinic, however, the detailed mechanism remains unclear. Objective: This research aims to comprehensively and systematically elucidate the mechanisms of EA treatment at the acupoints of Zusanli (ST36) and Baihui (GV20) on ischemic stroke. Methods: In this study, EA was performed twice at onset of reperfusion and 20 h after reperfusion following cerebral ischemia-reperfusion (I/R) in middle cerebral artery occlusion (MCAO) rats, and transcriptomic changes of various molecules in ischemic hippocampal neurons of rats in Sham, I/R and EA groups were detected by RNA-Sequencing (RNA-Seq). Results: Thus, we detected 18 significantly different genes related to atherosclerosis (AS), with their functions associated with lipid metabolism, thrombosis, monocytes and vascular smooth muscle cells. And, we detected 10 significantly different genes related to oxidative stress and apoptosis and 10 significantly different genes related to calcium overload and excitatory amino acids release. Also, we detected 19 significantly different genes related to blood-brain barrier (BBB) and 22 significantly different genes related to inflammatory response. Conclusion: In conclusion, EA can play a role in treating ischemic stroke through a variety of mechanisms, affecting atherosclerosis, oxidative stress, apoptosis, calcium overload, excitatory amino acids release, blood-brain barrier (BBB) and inflammatory response.