Chronic exposure to low-dose ionizing radiation is associated with an increased risk of cardiovascular disease. Alteration in energy metabolism has been suggested to contribute to radiation-induced heart pathology, mitochondrial dysfunction being a hallmark of this disease. The goal of this study was to investigate the regulatory role of acetylation in heart mitochondria in the long-term response to chronic radiation. ApoE-deficient C57Bl/6J mice were exposed to low-dose-rate (20 mGy/day) gamma radiation for 300 days, resulting in a cumulative total body dose of 6.0 Gy. Heart mitochondria were isolated and analyzed using quantitative proteomics. Radiation-induced proteome and acetylome alterations were further validated using immunoblotting, enzyme activity assays, and ELISA. In total, 71 proteins showed peptides with a changed acetylation status following irradiation. The great majority (94%) of the hyperacetylated proteins were involved in the TCA cycle, fatty acid oxidation, oxidative stress response and sirtuin pathway. The elevated acetylation patterns coincided with reduced activity of mitochondrial sirtuins, increased the level of Acetyl-CoA, and were accompanied by inactivation of major cardiac metabolic regulators PGC-1 alpha and PPAR alpha. These observations suggest that the changes in mitochondrial acetylation after irradiation is associated with impairment of heart metabolism. We propose a novel mechanism involved in the development of late cardiac damage following chronic irradiation.