Updated project metadata. Cytochrome c (Cytc) is essential for mitochondrial respiration and apoptosis. Tissue specific post-translational modifications of Cytc play an important regulatory role in these processes. Here, we describe a new acetylation site, lysine 39, which was mapped in ischemic porcine skeletal muscle. Recombinant acetylmimetic K39Q protein demonstrated increased cytochrome c oxidase (COX) activity and decreased caspase-3 and cardiolipin peroxidase activities. Cytc double knockout cells expressing acetylmimetic K39Q Cytc showed an increase in mitochondrial respiration, mitochondrial membrane potential, and mitochondrial ROS production and a decrease in cell death. These results are discussed in the context of X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that lysine 39 acetylation is an adaptive response allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.