Background The activation of extracellular signal-re gulated kinase1/2 (ERK 1/2 ) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mK ATP ) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mK ATP and ERK 1/2 in the delayed p rotection mechanism of anoxic preconditioning (APC). Methods An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptopro pionylglycine (MPG), and PD98059] were used to modulate mK ATP and ERK 1/2 activation. Cellular injury was evaluated by mea suring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK 1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mK ATP open er). Results Cell viability and SOD activity in the APC [(81.9±11.4)%, (13.6 ± 3.7) U/L] and diazoxide [(79.2±12.4)%, (16.5±4.6) U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42.2±7.3)%, (8.8±2. 8) U/L] group (all P<0.01). LDH activity in the APC group [(101.9±18.9) U/L] and diazoxide group [(97.5±17.7) U/L] was significantly lower than in the A/R group [(250.5±43.6) U/L] (all P<0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK 1/2 activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP (an mK ATP blocker) and by MPG (a free radical scavenger). In addition, the ERK 1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. Conclusion mK ATP may mediate ERK 1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes. Background The activation of extracellular signal-re gulated kinase 1/2 (ERK 1/2) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mK ATP) as both a trigger and an end effector of acute and delayed protection of IPC. Therefore, this study was undertaken to elucidate the relationship between mK ATP and ERK 1/2 in the delayed p rotection mechanism of anoxic preconditioning (APC ). Methods An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [diazoxide, 5-hydroxydecanoate (5-HD), 2-mercaptoproionylglycine (MPG), and PD98059] were used to modulate mK ATP and ERK 1/2 activation. Cellular injury was evaluated by mea suring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species nd the activation of ERK 1/2 were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mK ATP open er). Results Cell viability and SOD activity in the APC [(81.9 ± 11.4)%, ( 13.2 ± 3.7) U / L] and diazoxide [(79.2 ± 12.4)%, (16.5 ± 4.6) U / L] groups were significantly higher than in the anoxia / reoxygenation (A / R) (8.8 ± 2.8) U / L] group (all P <0.01). LDH activity in the APC group [(101.9 ± 18.9) U / L] and diazoxide group [(97.5 ± 17.7) U / L] was Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK 1/2 activation. But the effects of APC and diazoxide were lower than in the A / R group [(250.5 ± 43.6) U / L] (all P <0.01) remarkedly attenuated by 5-HP (an mK ATP blocker) and by MPG (a free radical scavenger). In addition, the ERK 1/2 inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. Conclusion mK ATP may mediateERK 1/2 activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes.