Background <p>The role of reactive oxygen species (ROS) in cancer is debated. One main antioxidant enzyme is mitochondrial superoxide dismutase (SOD2) which has been shown to influence tumor initiation and metastatic progression in several cancer types.</p> Methods <p>To investigate the impact of <i>Sod2</i> deletion on pancreatic cancer biology and metabolism, we used CRISPR/Cas9 gene editing to generate 3 independent <i>Sod2-</i>deficient cell lines from murine Kras^G12D pancreatic cancer cell lines and analyzed them for proliferation, colony forming, mitochondrial respiration and RNA expression. In addition, mass spectrometry and isotope tracing were performed.</p> Results <p>Proliferation and wound healing capacity were significantly impaired in <i>Sod2</i> deficient cell lines. Myc levels were significantly elevated in <i>Sod2</i>-deficient cells, and mitochondrial respiration was consecutively increased. This resulted in increased tolerance to glucose deprivation. Mechanistically, we detected a significantly reduced activity of succinate dehydrogenase (SDH) in <i>Sod2</i>-deficient cells. This resulted in increased peroxynitrite formation which was the cause of increased Myc activation.</p> Conclusions <p>These findings reveal that <i>Sod2</i> shapes cellular metabolism in pancreatic cancer through peroxynitrite formation and Myc activation.</p>