BACKGROUND - The B vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an antioxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and postreperfusion injury. METHODS AND RESULTS - Wistar rats were pretreated with either FA (10 mg/d) or placebo for 1 week and then underwent in vivo transient left coronary artery occlusion for 30 minutes with or without 90 minutes of reperfusion (total n=131; subgroups used for various analyses). FA (4.5×10 mol/L IC) pretreatment and global ischemia/reperfusion (30 minutes/30 minutes) also were performed in vitro (n=28). After 30 minutes of ischemia, global function declined more in controls than in FA-pretreated rats (ΔdP/dtmax, -878±586 versus -1956±351 mm Hg/s placebo; P=0.03), and regional thickening was better preserved (37.3±5.3% versus 5.1±0.6% placebo; P=0.004). Anterior wall perfusion fell similarly (-78.4±9.3% versus -71.2±13.8% placebo at 30 minutes), yet myocardial high-energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA pretreatment (ATP: control, 2740±58 nmol/g; ischemia, 947±55 nmol/g; ischemia plus FA, 1332±101 nmol/g; P=0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA pretreatment but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124±280 cpm/mg FA versus 5898±474 cpm/mg placebo; P=0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8±1.2% versus 60.3±4.1% placebo area at risk; P<0.002) and less contraction band necrosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positivity, superoxide, and nitric oxide synthase uncoupling. Infarct size declined similarly with 1 mg/d FA. CONCLUSIONS - FA pretreatment blunts myocardial dysfunction during ischemia and ameliorates postreperfusion injury. This is coupled to preservation of high-energy phosphates, reducing subsequent reactive oxygen species generation, eNOS-uncoupling, and postreperfusion cell death.