Despite advances in cardioprotection, new therapeutic strategies capable of preventing ischemia-reperfusion injury of patients are still needed. Here, we discover that sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA2) phosphorylation at serine 663 is a clinical and pathophysiological event of cardiac function. Indeed, the phosphorylation level of SERCA2 at serine 663 is increased in ischemic hearts of patients and mouse. Analyses on different human cell lines indicate that preventing serine 663 phosphorylation significantly increases SERCA2 activity and protects against cell death, by counteracting cytosolic and mitochondrial Ca²⁺ overload. By identifying the phosphorylation level of SERCA2 at serine 663 as an essential regulator of SERCA2 activity, Ca²⁺ homeostasis and infarct size, these data contribute to a more comprehensive understanding of the excitation/contraction coupling of cardiomyocytes and establish the pathophysiological role and the therapeutic potential of SERCA2 modulation in acute myocardial infarction, based on the hotspot phosphorylation level of SERCA2 at serine 663 residue.