Acute hyperglycemia co-presenting with myocardial infarctions is associated with poor prognosis. Higher oxidative stress and subsequent activation of non-oxidative glucose utilizing pathways (NOGP), i.e. hexosamine biosynthetic pathway (HBP), polyol pathway, advanced glycation end products (AGEs) and PKC have been implicated as potential mediators of this process. Hyperglycemia-induced oxidative stress can also result in dysregulation of the ubiquitin-proteasome system (UPS) that removes misfolded proteins. There are conflicting data whether increased/decreased UPS is detrimental with hyperglycemia and/or in response to ischemia-reperfusion. For example, we found cardioprotection with attenuated UPS activation following ischemia-reperfusion under hyperglycemic conditions, suggesting that increased UPS activity elicits damaging effects on the heart. We recently established that benfotiamine (BFT)-vitamin B1 analog- improved cardiac function under similar experimental conditions by shunting flux away from the NOGP and lowering myocardial oxidative stress. In light of this, we hypothesized that BFT acts as a novel cardioprotective agent by diminishing myocardial oxidative stress and UPS activity in response to ischemia-reperfusion under acute hyperglycemic conditions. Hearts were excised from male Wistar rats and perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± 100 µM BFT or 5 µM/L lactacystin (a specific UPS inhibitor) added during the first 20 min of the 60 min of reperfusion. In parallel experiments with similar treatments, we determined infarct sizes following 20 min regional ischemia and 120 min reperfusion. The data reveal that BFT enhanced functional recovery of LVDP at baseline (11 mM glucose) and hyperglycemia (33 mM) conditions with the highest dose (100 μM) triggering a robust effect early during the reperfusion period, reaching ~71.1 ± 5.9% (p<0.01 vs. respective control) under hyperglycemic perfusion conditions. BFT also reduced infarct sizes to 39.0 ± 2.5% and 49.7 ± 7.4% versus 52.3 ± 2.8% and 74.5 ± 4.9% at baseline and hyperglycemic perfusions, respectively. Likewise, lactacystin administration improved LVDP recovery and decreased infarct sizes following ischemia-reperfusion under hyperglycemic perfusion conditions. BFT treatment also attenuated myocardial oxidative stress and protein carbonylation, and blunted UPS activity. This study demonstrates that BFT may exert its cardioprotective effects by diminishing myocardial oxidative stress and UPS activity following ischemia-reperfusion under acute hyperglycemic conditions. Our findings suggest that the UPS may be a unique therapeutic target to treat ischemic heart disease in individuals that present with stress-induced, acute hyperglycemia.