Introduction. Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). EETs are formed from arachidonic acid during myocardial ischemia, and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. Hypothesis. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size after regional myocardial ischemia reperfusion injury in vivo. Methods. Male C57BL\6J wild-type (WT) or sEH knockout (sEHKO) mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. WT mice were injected intraperitoneal with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min prior to LCA occlusion or during ischemia 10 min prior to reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min prior to reperfusion. The EET antagonist 14,15-EEZE was given intravenously 15 min prior to reperfusion. Area-at-risk (AAR) and infarct size (I) were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and infarct size was expressed as I/AAR. Results. Infarct size was significantly reduced in animals treated with AUDA-BE or 14,15-EET independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Conclusion. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia reperfusion injury.