SOD2 is the primary antioxidant enzyme neutralizing •O2− in mitochondria. Cardiac-specific SOD2 overexpression (SOD2-tg) induces supernormal function and cardiac hypertrophy in the mouse heart. However, the reductive stress imposed by SOD2 overexpression results in protein aggregation of SOD2 pentamers and differential hyperacetylation of SOD2 in the mitochondria and cytosol. Here, we studied SOD2 acetylation in SOD2-tg and wild-type mouse hearts. LC-MS/MS analysis indicated the presence of four acetylated lysines in matrix SOD2 and nine acetylated lysines in cytosolic SOD2 from the SOD2-tg heart. However, only one specific acetylated lysine residue was detected in the mitochondria of the wild-type heart, which was consistent with Sirt3 downregulation in the SOD2-tg heart. LC-MS/MS further detected hyperacetylated SOD2 with a signaling peptide in the mitochondrial inner membrane and matrix of the SOD2-tg heart, indicating partial arrest of the SOD2 precursor in the membrane during translocation into the mitochondria. Upregulation of HSP 70 and cytosolic HSP 60 enabled the translocation of excess SOD2 into mitochondria. In vitro acetylation of matrix SOD2 with Ac2O deaggregated pentameric SOD2, restored the profile of cytosolic SOD2 hyperacetylation, and decreased matrix SOD2 activity. As revealed by 3D structure, acetylation of K89, K134, and K154 of cytosolic SOD2 induces unfolding of the tertiary structure and breaking of the salt bridges that are important for the quaternary structure, suggesting that hyperacetylation and HSP 70 upregulation maintain the unfolded status of SOD2 in the cytosol and mediate the import of SOD2 across the membrane. Downregulation of Sirt3, HSP 60, and presequence protease in the mitochondria of the SOD2-tg heart promoted protein misfolding that led to pentameric aggregation.