Mitochondria manganese superoxide dismutase (SOD2) is an important antioxidant enzyme, deficiency of which is associated with various human diseases. The known primary regulation of SOD2 is through transcriptional activation. Here, we report that SOD2 is acetylated at Lys 68 and that this acetylation decreases SOD2 activity. Mitochondrial deacetylase SIRT3 binds to, deacetylates and activates SOD2. Increase of reactive oxygen species (ROS) levels stimulates SIRT3 transcription, leading to SOD2 deacetylation and activation. SOD2-mediated ROS reduction is synergistically increased by SIRT3 co-expression, but is cancelled by SIRT3 depletion. These results reveal a new post-translational regulation of SOD2 by means of acetylation and SIRT3-dependent deacetylation in response to oxidative stress.