The sulfonylurea receptor-2 (SUR2) is a subunit of ATP-sensitive potassium channels (K_ATP) in heart. Mice with the SUR2 gene disrupted (SUR2m) are constitutively protected from ischemia-reperfusion (I/R) cardiac injury. This was surprising because K_ATP, either sarcolemmal or mitochondrial or both, are thought to be important for cardioprotection. We hypothesized that SUR2m mice have an altered mitochondrial phenotype that protects against I/R. Mitochondrial membrane potential (ΔΨ_m), tolerance to Ca²⁺ load, and reactive oxygen species (ROS) generation were studied by fluorescence-based assays, and volumetric changes in response to K⁺ were measured by light scattering in isolated mitochondria. For resting SUR2m mitochondria compared with wild type, the ΔΨ_m was less polarized (46.1 ± 0.4 vs. 51.9 ± 0.6%), tolerance to Ca²⁺ loading was increased (163 ± 2 vs. 116 ± 2 μM), and ROS generation was enhanced with complex I [8.5 ± 1.2 vs. 4.9 ± 0.2 arbitrary fluorescence units (afu)/s] or complex II (351 ± 51.3 vs. 166 ± 36.2 afu/s) substrates. SUR2m mitochondria had greater swelling in K⁺ medium (30.2 ± 3.1%) compared with wild type (14.5 ± 0.6%), indicating greater K⁺ influx. Additionally, ΔΨ_m decreased and swelling increased in the absence of ATP in SUR2m, but the sensitivity to ATP was less compared with wild type. When the mitochondria were subjected to hypoxia-reoxygenation, the decrease in respiration rates and respiratory control index was less in SUR2m. ΔΨ_m maintenance in the SUR2m intact myocytes was also more tolerant to metabolic inhibition. In conclusion, the cardioprotection observed in the SUR2m mice is associated with a protected mitochondrial phenotype resulting from enhanced K⁺ conductance that partially dissipated ΔΨ_m. These results have implications for possible SUR2 participation in mitochondrial K_ATP.