Accumulation of superoxide radicals leads to disrupted redox signaling and oxidative damage. The primary extracellular scavenger of superoxide is extracellular superoxide dismutase (SOD3), a crucial enzyme in maintaining antioxidant status and proper immune function. SOD3 distribution to the extracellular matrix is determined by the presence of a C-terminal heparin-binding domain (HBD). This region can be removed through intracellular proteolytic processing by furin. Cleavage of the HBD has been shown to be modulated by post-translational cysteine redox status, regulating the secretion of SOD3. Interestingly, other members of the SOD family, SOD1 and SOD2, are known to be inhibited by lysine acetylation, a metabolically linked post-translational modification (PTM) that can alter protein structure, function, and localization. Yet, no reports describe the effect of acetylation on SOD3. Here, immunoblotting and mass spectrometry (MS) were used to quantify the global and site-specific acetylation of recombinant human SOD3. Interestingly, a predicted and targeted parallel reaction monitoring (PRM) MS-based approach was necessary to identify lysine acetylation within the C-terminal HBD of SOD3. Acetylation was found to prevent furin cleavage with no impact on SOD3 activity. Our results also reveal that SOD3 is robustly deacetylated by NAD+-dependent sirtuins (SIRT1 and SIRT3), with moderate activity against K220 and high activity against K211 and K212 in the HBD furin cleavage region. These sites of acetylation have not been previously reported, likely due to the peptide's unique hydrophilic nature. Overall, our findings reveal that sirtuin-directed deacetylation of SOD3 restored furin cleavage, defining an important link between redox homeostasis and acetylation-directed metabolic regulation of extracellular oxidative stress.