It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6 ± 5.7% vs. 59.4 ± 6.1%; p < 0.05) and decreased cardiac troponin I release (25.4 ± 3.0 vs. 4.7 ± 1.9 ng/ml; p < 0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 μM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels. © 2006 Elsevier Inc. All rights reserved.