Background/Objectives: Ischemic heart disease remains a leading cause of morbidity and mortality worldwide, accompanied by a major decline in local myocardial pH. However, the mechanisms of pH regulation and the homeostasis of H⁺ neutralizing buffers, such as HCO₃⁻, in cardiomyocytes remain incompletely understood. We identified a solute carrier, Slc26a6, in mouse and human hearts playing key roles in the regulation of cardiac pH, excitability, and contractility. Slc26a6 is an acid loader, so we hypothesized that ablation of Slc26a6 may protect the heart from ischemia/reperfusion (I/R) injury. Methods: The I/R model was generated using wild type (WT) and Slc26a6 knockout (Slc26a6^−/−) mice. Multidisciplinary in vivo, in vitro, and ex vivo approaches were used, including echocardiography, electrophysiology, hemodynamic monitoring, fluorescence microscopy, histochemistry, and cellular Ca²⁺ transients, sarcoplasmic reticulum Ca²⁺ load, and sarcomere shortening were recorded. Results: Troponin I level was lower in Slc26a6^−/− I/R mice. Slc26a6^−/− mice showed better systolic and diastolic function, reduced collagen deposition, and reduced infarct size compared to that of WT mice. Cellular experiments in measurement of sarcomere shortening, Ca²⁺ transients, and sarcoplasmic reticulum Ca²⁺ load in cardiomyocytes from the infarct zone supported the in vivo findings, demonstrating better single cell function in Slc26a6^−/− compared to WT mice. Ex vivo pH_i measurement showed elevated pH_i in Slc26a6^−/− mouse heart. Conclusions: Ablation of Slc26a6 protects the heart from I/R injury, suggesting the importance of Cl⁻/HCO₃⁻ exchange in cardiac pH regulation and I/R injury. The elevated pH_i in Slc26a6^−/− mouse heart may counterbalance the effects of the myocardium acidosis resulting from ischemia.