Background Supplemental oxygenation of the standard hypothermic machine perfusion (HMP) circuit has the potential to invoke favorable changes in metabolism, optimizing cadaveric organs before transplantation. Methods Eight pairs of porcine kidneys underwent 18 hours of either oxygenated (HMP/O 2 ) or aerated (HMP/Air) HMP in a paired donation after circulatory death model of transplantation. Circulating perfusion fluid was supplemented with the metabolic tracer universally labeled glucose. Perfusate, end-point renal cortex, and medulla samples underwent metabolomic analysis using 1-dimension and 2-dimension nuclear magnetic resonance experiments in addition to gas chromatography-mass spectrometry. Analysis of 13 C-labeled metabolic products was combined with adenosine nucleotide levels and differences in tissue architecture. Results Metabolomic analysis revealed significantly higher concentrations of universally labeled lactate in the cortex of HMP/Air versus HMP/O 2 kidneys (0.056 mM vs 0.026 mM, P < 0.05). Conversely, newly synthesized [4,5- 13 C] glutamate concentrations were higher in the cortex of HMP/O 2 kidneys inferring relative increases in tricarboxylic acid cycle activity versus HMP/Air kidneys (0.013 mmol/L vs 0.003 mmol/L, P < 0.05). This was associated with greater amounts of adenoside triphosphate in the cortex HMP/O 2 versus HMP/Air kidneys (19.8 mmol/mg protein vs 2.8 mmol/mg protein, P < 0.05). Improved flow dynamics and favorable ultrastructural features were also observed in HMP/O 2 kidneys. There were no differences in thiobarbituric acid reactive substances and reduced glutathione levels, tissue markers of oxidative stress, between groups. Conclusions The supplementation of perfusion fluid with high-concentration oxygen (95%) results in a greater degree of aerobic metabolism versus aeration (21%) in the nonphysiological environment of HMP, with reciprocal changes in adenoside triphosphate levels.