Tracking biochemical events in vivo is one of the most ambitious goals of modern physiology. Cardiac magnetic resonance (MRI) with hyperpolarized 13C-pyruvate is a promising new technique for the assessment of myocardial energy substrate metabolism, in intact hearts. We tested the effectiveness of this technique to quantify myocardial pyruvate uptake and the formation of its metabolites lactate (reduction) and CO2/bicarbonate (full oxidation in the Krebs cycle) in a pig model of acute ischemia/reperfusion. Seven pigs were chronically instrumented with a pneumatic occluder placed around the left anterior descending coronary artery. After recovery, they were subjected to MRI with a 3T scanner and a 13C quadrature birdcage coil. 20 ml of 13C-pyruvate (230 mM) were hyperpolarized using Dynamic Nuclear Polarization and injected intravenously at rest, during coronary occlusion (10 minutes) and at 5 minutes of reperfusion. During occlusion, we found a 17% decrease of 13C-lactate and a 62% decrease of 13C-bicarbonate in the area at risk as compared with remote myocardium. In the same area, the 13C-lactate signal increased by 27% during reperfusion, consistent with the enhanced pyruvate reduction in the post-ischemic phase, while 13C-bicarbonate was found persistently reduced, consistent with the expected presence of post-ischemic depression of the oxidative metabolism. In conclusion, 13C-pyruvate MRI is sufficiently sensitive to detect transient changes in regional myocardial metabolism in an in vivo model of ischemia-reperfusion.