Despite years of experimental and clinical research, myocardial ischemia-reperfusion (IR) remains an important cause of cardiac morbidity and mortality. The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated as a key mediator of reperfusion injury. Activation of NF-kappaB is dependent upon the phosphorylation of its inhibitor, IkappaBalpha, by the specific inhibitory kappaB kinase (IKK) subunit, IKKbeta. We hypothesized that specific antagonism of the NF-kappaB inflammatory pathway through IKKbeta inhibition reduces acute myocardial damage following IR injury. C57BL/6 mice underwent left anterior descending (LAD) artery ligation and release in an experimental model of acute IR. Bay 65-1942, an ATP-competitive inhibitor that selectively targets IKKbeta kinase activity, was administered intraperitoneally either prior to ischemia, at reperfusion, or 2 h after reperfusion. Compared with untreated animals, mice treated with IKKbeta inhibition had significant reduction in left ventricular infarct size. Cardiac function was also preserved following pretreatment with IKKbeta inhibition. These findings were further associated with decreased expression of phosphorylated IkappaBalpha and phosphorylated p65 in myocardial tissue. In addition, IKKbeta inhibition decreased serum levels of TNF-alpha and IL-6, two prototypical downstream effectors of NF-kappaB activity. These results demonstrate that specific IKKbeta inhibition can provide both acute and delayed cardioprotection and offers a clinically accessible target for preventing cardiac injury following IR.