Ischemia-reperfusion (I/R) injury is a major cause of morbidity and mortality worldwide. AMP-activated protein kinase (AMPK) is an energy sensor that regulates metabolic homeostasis. A growing body of literature has shown that AMPK activation exerts protective effects against I/R injury in heart, brain, kidney, liver, lung and intestine. In this review, we first reveal the mechanisms underlying the protective effects of AMPK activation against I/R injury in preclinical studies. We found that AMPK activation attenuates I/R injury via regulation of energy metabolism, oxidative stress, mitochondrial function, autophagy, inflammatory response, and endoplasmic re-ticulum stress. Then, current therapeutic strategies (e.g., metformin, adiponectin) used to ameliorate I/R injury by modulating AMPK activity are reviewed in detail. Collectively, pharmacological activation of AMPK may hold a unique therapeutic potential in the prevention and attenuation of I/R injury.