Here we found that low-concentration of perifosine, an Akt inhibitor, surprisingly protected cardiomyocytes from oxygen glucose deprivation (OGD)/re-oxygenation. In H9c2 cardiomyocytes, non-cytotoxic perifosine (0.1-0.5 mu M) suppressed OGD/re-oxygenation-induced reactive oxygen species (ROS) production, p53 mitochondrial translocation and cyclophilin D complexation, as well as mitochondrial membrane potential (MMP) reduction. Molecularly, perifosine activated AMP-activated kinase (AMPK) signaling to increase intracellular NADPH (nicotinamide adenine dinucleotide phosphate) content in H9c2 cells. On the other hand, AMPK inhibition by AMPK alpha 1 shRNA-knockdown in H9c2 cells significantly reduced perifosine-induced NADPH production, and alleviated perifosine-mediated antioxidant and cytoprotective activities against OGD/re-oxygenation. In primary murine cardiomyocytes, perifosine similarly activated AMPK signaling, and offered significant protection against OGD/re-oxygenation, which was largely attenuated with siRNA knockdown of AMPK alpha 1. We demonstrate an unexpected function of perifosine (low-concentration) in protecting cardiomyocytes from OGD/re-oxygenation. (C) 2015 Elsevier Inc. All rights reserved.