ATP-sensitive potassium channels found in both the sarcolemma (sarcK_ATP) and mitochondria (mitoK_ATP) of cardiomyocytes are important mediators of cardioprotection during ischemic heart disease. Sulfonylurea receptor isoforms (SUR2), encoded by Abcc9, an ATP-binding cassette family member, form regulatory subunits of the sarcK_ATP channel and are also thought to regulate mitoK_ATP channel activity. A short-form splice variant of SUR2 (SUR2A-55) was previously shown to target mitochondria and display diaxoxide and ATP insensitive K_ATP activity when co-expressed with the inward rectifier channels Kir6.2 and Kir6.1. We hypothesized that mice with cardiac specific overexpression of SUR2A-55 would mediate cardioprotection from ischemia by altering mitoK_ATP properties. Mice overexpressing SUR2A-55 (TG^SUR2A-55) in cardiomyocytes were generated and showed no significant difference in echocardiographic measured chamber dimension, percent fractional shortening, heart to body weight ratio, or gross histologic features compared to normal mice at 11–14 weeks of age. TG^SUR2A-55 had improved hemodynamic functional recovery and smaller infarct size after ischemia reperfusion injury compared to WT mice in an isolated hanging heart model. The mitochondrial membrane potential of TG^SUR2A-55 mice was less sensitive to ATP, diazoxide, and Ca²⁺ loading. These data suggest that the SUR2A-55 splice variant favorably affects mitochondrial function leading to cardioprotection. These data support a role for the regulation of mitoK_ATP activity by SUR2A-55.