Acute myocardial infarction (AMI) is the most severe form of coronary heart disease caused by ischemia and hypoxia. The study is aimed at investigating the role of neuropeptides and the mechanism of electroacupuncture (EA) in acute myocardial infarction (AMI) treatment. Compared with the normal population, a significant increase in substance P (SP) was observed in the serum of patients with AMI. PGI2 expression was increased in the SP-treated AMI mouse model, and TXA2 expression was decreased. And PI3K pathway-related genes, including Pik3ca, Akt, and Mtor, were upregulated in myocardial tissue of SP-treated AMI patients. Human cardiomyocyte cell lines (HCM) treated with SP increased mRNA and protein expression of PI3K pathway-related genes (Pik3ca, Pik3cb, Akt, and Mtor). Compared to MI control and EA-treated MI rat models, Myd88, MTOR, Akt1, Sp, and Irak1 were differentially expressed, consistent with in vivo and in vitro studies. EA treatment significantly enriched PI3K/AKT signaling pathway genes within MI-associated differentially expressed genes (DEGs) according to Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, it was confirmed by molecular docking analysis that PIK3CA, AKT1, and mTOR form stable dockings with neuropeptide SP. PI3K/AKT pathway activity may be affected directly or indirectly by EA via SP, which corrects the PGI2/TXA2 metabolic imbalance in AMI. MI treatment is now better understood as a result of this finding.