This study investigates the protective effects of inhibiting the mitochondrial permeability transition pore (mPTP) and lipid peroxidation (LIPOX) against ischemia-reperfusion (I/R) injury in mice. The research highlights that both Ca²⁺ and reactive oxygen species (ROS) contribute to mitochondrial dysfunction during I/R injury, but they act through different mechanisms. While Ca²⁺ primarily triggers mPTP opening, leading to mitochondrial swelling and necrotic death, ROS can cause mitochondrial damage through LIPOX, a process linked to ferroptosis. However, subtoxic levels of both Ca²⁺ and ROS can synergistically induce mPTP opening, suggesting a complex interplay between these triggers. The study demonstrates that dual inhibition of mPTP and LIPOX is significantly more protective against I/R injury than targeting either pathway alone. Inhibitors such as cyclosporine A (CsA) for mPTP and MitoQ for LIPOX were shown to reduce infarct size in mice. These findings suggest that targeting both pathways could be an effective therapeutic strategy for mitigating I/R injury. The research also reveals that ROS can sensitize the mPTP to Ca²⁺, potentially through modifications of cyclophilin D (CypD). This sensitization is independent of CypD, indicating that other mechanisms may be involved. Furthermore, the study shows that the synergistic effect of Ca²⁺ and ROS on mPTP opening persists even in the absence of CypD, suggesting that multiple pathways contribute to mPTP activation. Overall, the study underscores the importance of understanding the distinct and overlapping roles of Ca²⁺ and ROS in mitochondrial dysfunction during I/R injury. The findings support the development of therapeutic approaches that target both mPTP and LIPOX to improve outcomes in patients with I/R injury.This study investigates the protective effects of inhibiting the mitochondrial permeability transition pore (mPTP) and lipid peroxidation (LIPOX) against ischemia-reperfusion (I/R) injury in mice. The research highlights that both Ca²⁺ and reactive oxygen species (ROS) contribute to mitochondrial dysfunction during I/R injury, but they act through different mechanisms. While Ca²⁺ primarily triggers mPTP opening, leading to mitochondrial swelling and necrotic death, ROS can cause mitochondrial damage through LIPOX, a process linked to ferroptosis. However, subtoxic levels of both Ca²⁺ and ROS can synergistically induce mPTP opening, suggesting a complex interplay between these triggers. The study demonstrates that dual inhibition of mPTP and LIPOX is significantly more protective against I/R injury than targeting either pathway alone. Inhibitors such as cyclosporine A (CsA) for mPTP and MitoQ for LIPOX were shown to reduce infarct size in mice. These findings suggest that targeting both pathways could be an effective therapeutic strategy for mitigating I/R injury. The research also reveals that ROS can sensitize the mPTP to Ca²⁺, potentially through modifications of cyclophilin D (CypD). This sensitization is independent of CypD, indicating that other mechanisms may be involved. Furthermore, the study shows that the synergistic effect of Ca²⁺ and ROS on mPTP opening persists even in the absence of CypD, suggesting that multiple pathways contribute to mPTP activation. Overall, the study underscores the importance of understanding the distinct and overlapping roles of Ca²⁺ and ROS in mitochondrial dysfunction during I/R injury. The findings support the development of therapeutic approaches that target both mPTP and LIPOX to improve outcomes in patients with I/R injury.