In the clinical setting, bicarbonate is often used to correct acidosis arising from accumulated CO2/HCO3- during ischemia. We observed that HL-1 cardiomyocytes exposed to increased [CO2/HCO3-] had more cell death after hypoxia/reoxygenation (H/R) and Langendorff-perfused rat hearts had larger infarcts after ischemia/reperfusion (I/R). In addition to buffering pH, the CO2/HCO3- pair possess an underappreciated redox activity that may contribute to injury. In order to study the effects of high CO2/HCO3- during ischemic injury independent of pH, we clamped pH using HEPES and used the mouse cardiomyocyte HL-1 cell line and isolated perfused rat hearts. HL-1 cells exposed to 10% CO2/HCO3- had no damage under basal conditions but developed exaggerated protein carbonylation and cell death after H/R. In Langendorff-perfused rat hearts, 10% CO2 was well tolerated during baseline conditions but resulted in increased protein carbonylation, cell death and larger infarcts after I/R. We hypothesized that the increased oxidative damage to proteins could be due to mitochondrial dysfunction with greater ROS production, diminished proteasomal degradation of oxidized proteins, or impaired autophagic clearance of damaged mitochondria and oxidized protein aggregates. There was no differential effect of CO2 on mitochondrial morphology or proteasomal activity in HL-1 cells. In mitochondria isolated from perfused hearts subjected to I/R under low and high CO2 conditions, there was no difference in ROS production or oxidized protein content, suggesting mitochondrial damage was not affected by CO2 level. Examination of autophagy in HL-1 cells exposed to high CO2 during H/R revealed higher LC3-II and lower p62 content. In hearts, changes in LC3-II were inconsistent; however, we detected less p62 protein, less mitochondria-associated Beclin1, and significantly more LC3 mRNA in hearts exposed to 10% CO2 during I/R. Taken together, these findings suggest that 10% CO2 affects autophagy, which could explain the accumulation of oxidatively damaged proteins. These findings point to a protective role for autophagic clearance of oxidized protein aggregates during I/R injury that may be adversely impacted by bicarbonate therapy.