We investigated the implication of AMP-activated protein kinase (AMPK) in melatonin-induced protection in a renal ischemia/reperfusion (I/R) model. Animals were divided into four groups: rats of the sham group were not subjected to renal I/R. Rats in the I/R group received the vehicle 30 min before renal ischemia for 1 h followed by reperfusion for 6 h. Rats in the Mel+I/R group received melatonin (10 mg/kg) 30 min before the onset of I/R. Rats in the Mel+I/R+araA group received araA (AMPK inhibitor, 100 μg/kg per min for 10 min) just before melatonin administration. The results showed that melatonin treatment induced a significant improvement in renal function, morphology, and antioxidant status, as well as Akt pathway activation. It also decreased cytolysis and endoplasmic reticulum stress. Inhibition of AMPK by araA administration completely abolished the effects of melatonin in regard to the above parameters except for Akt and some of its downstream target molecules. These results demonstrate that the effects of melatonin on renal I/R are, for the most part, linked to AMPK activation, with the exception of the Akt pathway, which seems to be independent of AMPK.