Recent studies have implied that activation of AMP-dependent protein kinase (AMPK) could protect myocardial cells from oxygen glucose deprivation-re-oxygenation (OGD/R). The aim of the present study is to test whether GSK621, a novel and direct AMPK activator, could exert myocardial cell protection against OGD/R. We show that in AC16 human myocardial cells and primary murine myocardiocytes GSK621 dose-dependently activated AMPK signaling. GSK621 pretreatment potently inhibited OGD/R-induced viability reduction, cell death and apoptosis in AC16 cells and primary myocardiocytes. Furthermore, GSK621 attenuated OGD/R-induced reactive oxygen species production and oxidative injury in the myocardial cells. AMPKα1 knockdown (via targeted shRNA), knockout (via a CRISPR/Cas9 construct) or dominant negative mutation (T172A) not only blocked GSK621-induced AMPK activation, but also nullified GSK621-mediated myocardial cell protection against OGD/R. Further studies demonstrated that GSK621 activated AMPK downstream Nrf2 signaling. Contrarily, Nrf2 silencing by targeted shRNAs almost abolished GSK621-induced anti-OGD/R myocardial cell protection. We conclude that GSK621 protects myocardial cells from OGD/R through activation of AMPK-dependent signaling.