Erythropoietin (EPO) has protective effects on myocardial infarction (MI). This study was to test whether EPO administrated after MI is as effective as EPO administrated before MI and to determine the role of AMP-activated protein kinase (AMPK) in the cardioprotective effects of EPO. Recombinant human EPO (5000 IU/kg) was intraperitoneally injected 12 hours before MI (pretreatment) or 30 minutes after MI (posttreatment). The levels of serum enzymes were assayed at 24 and 72 hours after MI. The infarct size was determined by nitro blue tetrazolium staining. The microarchitecture damages were evaluated by electron microscopy. EPO receptor messenger RNA and protein levels were determined by RT-PCR and immunohistochemistry, respectively. Activation of AMPK and nuclear factor kappa B was determined by immunoblotting. An AMPK-specific inhibitor, compound C, was used to inhibit AMPK activation in vivo. The authors found that both pretreatment and posttreatment of EPO successfully attenuated the serum enzyme levels, reduced the infarct size and ameliorated the microarchitecture damage. Moreover, both pretreatment and posttreatment of EPO decreased the EPO receptor messenger RNA and protein expressions, which were up-regulated in MI. More importantly, under MI conditions, EPO further increased the phosphorylation of AMPK and suppressed the activation of nuclear factor kappa B. Moreover, when AMPK was blocked by compound C, the cardioprotective effect of EPO was significantly attenuated (P < 0.01). Thus, this study demonstrates that both pretreatment and posttreatment of EPO are cardioprotective in an AMPK-dependent manners, providing the first evidence that the AMPK signaling pathway is involved in the cardioprotective effects of EPO.