Abstract: AIM To investigate the effects of punicalagin (PUN) on myocardial ischemia-reperfusion (MI/R) injury and underlying mechanisms. METHODS PUN30 mg/(kg·d) was intragastrically administered to male Sprague-Dawley rats for 7 d before operation. MI/R was induced by ligating the left anterior descending coronary artery for 30 min and subsequent reperfusion for 3 h. Cardiomyocyte apoptosis was determined by a terminal deoxynucleotidyl nick-end labeling (TUNEL) assay. The antioxidant enzyme superoxide dismutase (SOD) activities and malonaldialdehyde (MDA) levels in myocardial homogenates were determined spectrophotometrically. Outcome measures included cardiac functions, myocardial injury, oxidative stress and levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) protein. RESULTS PUN pretreatment conferred cardioprotective effects against MI/R injury by improving cardiac functions, limiting infarct size, reducing serum creatine kinase-MB and lactate dehydrogenase activities, and suppressing cardiomyocyte apoptosis. Moreover, PUN pretreatment inhibited I/R-induced myocardial oxidative stress as evidenced by decreased malonaldialdehyde formation and increased superoxide dismutase activity (both, P<0.05 or 0.01). Furthermore, PUN pretreatment increased AMPK phosphorylation in I/R hearts. AMPK inhibitor compound c blunted PUN-mediated cardioprotection against MI/R injury (P<0.05 or 0.01). CONCLUSION These results indicate that PUN pretreatment protects against I/R-induced myocardial injury via activation of AMPK.