Mitochondrial calcium (Ca²⁺) signals play a central role in cardiac homeostasis and disease. In healthy hearts, mitochondrial Ca²⁺ levels modulate oxidative metabolism to match ATP consumption. During ischemia/reperfusion (I/R) injury, pathologically high Ca²⁺ levels in the mitochondrial matrix trigger the opening of the mitochondrial permeability transition pore (mPTP), leading to mitochondrial swelling and cell death. Pharmacological and genetic approaches to regulate mitochondrial Ca²⁺ handling, such as modulating the mitochondrial Ca²⁺ uniporter (MCU) and sodium/Ca²⁺ exchanger (NCLX), represent promising therapeutic strategies to protect the heart from I/R injury. The review outlines the physiological role of mitochondrial Ca²⁺ signals, their alterations during I/R injury, and how these signals can be modulated to prevent cardiac damage. It highlights the importance of mechano-energetic coupling, where Ca²⁺ levels in the mitochondrial matrix are finely tuned by the balance between Ca²⁺ uptake via the MCU and Ca²⁺ extrusion via the NCLX. The structural and functional interaction between the sarcoplasmic reticulum (SR) and mitochondria is crucial for Ca²⁺ signal transmission to the mitochondrial matrix. During I/R injury, mitochondrial Ca²⁺ overload is a major driver of cardiac myocyte loss. Excessive Ca²⁺ release from the SR and oxidative stress during reperfusion trigger mPTP opening, leading to irreversible mitochondrial damage and cell death. The review discusses the role of mitochondrial reactive oxygen species (ROS) in I/R injury, including the concept of redox-optimized ROS balance and ROS-induced ROS release. Ischemic conditioning, a cardioprotective program induced by repeated brief episodes of coronary occlusion and reperfusion, is also explored. The review highlights the role of mitochondrial ATP-dependent K⁺ channels (mKATP) and connexin 43 (Cx43) in ischemic conditioning, and how modulation of these channels can reduce susceptibility to I/R injury. Finally, the review discusses strategies to modulate mitochondrial Ca²⁺ handling for cardioprotection, including pharmacological and genetic approaches to inhibit the MCU and enhance NCLX activity. The findings suggest that targeting mitochondrial Ca²⁺ handling can be a double-edged sword, but may be successful when tailored to specific pathological conditions. Further research is needed to better understand the fine-tuning of mitochondrial Ca²⁺ handling and its alterations in different pathological conditions.Mitochondrial calcium (Ca²⁺) signals play a central role in cardiac homeostasis and disease. In healthy hearts, mitochondrial Ca²⁺ levels modulate oxidative metabolism to match ATP consumption. During ischemia/reperfusion (I/R) injury, pathologically high Ca²⁺ levels in the mitochondrial matrix trigger the opening of the mitochondrial permeability transition pore (mPTP), leading to mitochondrial swelling and cell death. Pharmacological and genetic approaches to regulate mitochondrial Ca²⁺ handling, such as modulating the mitochondrial Ca²⁺ uniporter (MCU) and sodium/Ca²⁺ exchanger (NCLX), represent promising therapeutic strategies to protect the heart from I/R injury. The review outlines the physiological role of mitochondrial Ca²⁺ signals, their alterations during I/R injury, and how these signals can be modulated to prevent cardiac damage. It highlights the importance of mechano-energetic coupling, where Ca²⁺ levels in the mitochondrial matrix are finely tuned by the balance between Ca²⁺ uptake via the MCU and Ca²⁺ extrusion via the NCLX. The structural and functional interaction between the sarcoplasmic reticulum (SR) and mitochondria is crucial for Ca²⁺ signal transmission to the mitochondrial matrix. During I/R injury, mitochondrial Ca²⁺ overload is a major driver of cardiac myocyte loss. Excessive Ca²⁺ release from the SR and oxidative stress during reperfusion trigger mPTP opening, leading to irreversible mitochondrial damage and cell death. The review discusses the role of mitochondrial reactive oxygen species (ROS) in I/R injury, including the concept of redox-optimized ROS balance and ROS-induced ROS release. Ischemic conditioning, a cardioprotective program induced by repeated brief episodes of coronary occlusion and reperfusion, is also explored. The review highlights the role of mitochondrial ATP-dependent K⁺ channels (mKATP) and connexin 43 (Cx43) in ischemic conditioning, and how modulation of these channels can reduce susceptibility to I/R injury. Finally, the review discusses strategies to modulate mitochondrial Ca²⁺ handling for cardioprotection, including pharmacological and genetic approaches to inhibit the MCU and enhance NCLX activity. The findings suggest that targeting mitochondrial Ca²⁺ handling can be a double-edged sword, but may be successful when tailored to specific pathological conditions. Further research is needed to better understand the fine-tuning of mitochondrial Ca²⁺ handling and its alterations in different pathological conditions.