Background: Coronary artery disease is a leading public health problem. However, the mechanisms underlying mitochondrial damage remain unclear. The present study verified and explored the novel mechanisms underlying ischemic injury based on a metabolomic analysis. Methods: Mouse models of acute myocardial infarction were established, and serum samples were collected for targeted liquid chromatography with tandem mass spectrometry analysis. Based on metabolomic analyses, the N-methyl-d-aspartic acid receptor (NMDAR)–related calcium transporting signaling pathway was selected. Primary cardiomyocyte cultures were used, and N-methyl-d-aspartic acid (NMDA) was used as an agonist to confirm the role of NMDAR in ischemic injury. In addition, Bax, Bcl-2, mitochondrial calcium, potential, and mitochondrial reactive oxygen species accumulation were used to explore the role of NMDAR in mitochondrial damage–induced apoptosis. Results: Glutamate-related metabolism was significantly altered following in acute myocardial infarction. NMDA induces apoptosis under hypoxic conditions NMDAR was translocated to the mitochondrial-related membrane after activation, and its mitochondrial expression was significantly increased (p 0.05). Mitochondrial damage–induced apoptosis was significantly inhibited by a selective NDMAR antagonist (p 0.05), while Bax expression was remarkably decreased and Bcl-2 expression was increased (p 0.05). To further explore the mechanism of NMDAR, mitochondrial calcium, membrane potential, and reactive oxygen species were detected. With NMDAR inhibition under hypoxic conditions, mitochondrial morphology and function were preserved (p 0.05). Conclusions: Our metabolomic study identified NMDAR as a promising target. In conclusion, our study provides solid data for further studies of the role of NMDAR in cardiovascular diseases and a promising target to interfere with apoptosis in acute myocardial infarction.