Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3^−/− livers at 3 months of age. Livers of Sirt3^−/− mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3^−/− MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD^K122-R), increased MnSOD activity when expressed in MnSOD^−/− MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3^−/− MEFs with lenti-MnSOD^K122-R inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3^−/− livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.