Abstract Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCɛ). We recently identified aldehyde dehydrogenase 2 (ALDH2) as a PKCɛ substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo . ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCɛ knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCɛ and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCɛ, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCɛ knockout hearts. Our data suggest that ALDH2 is downstream of PKCɛ in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCɛ to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.