Aims: Mitochondrial Ca²⁺ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca²⁺ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca²⁺ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca²⁺ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α₁-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α₁-adrenoceptor (α₁-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca²⁺ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α₁-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca²⁺ overload. Innovation: Our data indicate that inhibition of α₁-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca²⁺ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α₁-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca²⁺ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879.