Myocardial ischemia-reperfusion injury (MIRI) has high morbidity and mortality worldwide. Increasing evidence has shown that electro-acupuncture (EA) plays a critical role in alleviating MIRI. The aim of this study is to investigate whether glutamatergic neurons in the lateral hypothalamus (LH) has vital effect on MIRI as well as the underlying mechanism during the EA pretreatment. The MIRI model was established by ligating the left anterior descending (LAD) coronary artery for 30 minutes followed by reperfusion for 2 hours. Chemogenetics, electrocardiogram (ECG) recording, ELISA, multi-channel physiology recording and immunofluorescence staining methods were combined to demonstrate that firing frequencies of neurons in the LH and expression of c-Fos decreased by EA pretreatment. Meanwhile, EA preconditioning significantly reduced the percentage of infarct size and the levels of cTnI and CK-MB were similar to inhibition of Gluergic neurons in LH, also attenuated morphology of myocardial tissue was induced by MIRI. However, activation of glutamatergic neurons in LH weakened the above effects of EA pretreatment.