We and others have reported that Rho-kinase plays an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. Studies have also demonstrated that the activation of Rho-kinase is reversed in ischemic preconditioning (IPC). However, the mechanisms by which Rho-kinase is increased in I/R and reversed in IPC are not thoroughly understood. In female Wistar rats, we created I/R by ligating the left anterior-descending branch of the coronary artery (LAD) for 30 min and releasing the ligature for 180 min. IPC rats underwent IPC (two cycles of 5-min ligation of the LAD and 5-min reflow) before I/R. IPC caused a significant increase in extracellular signal-regulated kinase (ERK)1/2 activity and reduced Rho-kinase activity and cardiomyocyte apoptosis (P<0.05 versus I/R). Administration of PD98059, an inhibitor of ERK-mitogen-activated protein kinase (MAPK), increased cardiomyocyte apoptosis, Caspase-3 activity and myocardial infarction size (P<0.05 versus IPC). Western-blot analysis showed that administration of PD98059 increased Rho-kinase activity. Treatment with fasudil, an inhibitor of Rho-kinase, reversed cell apoptosis caused by treatment with PD98059 in IPC. In addition, ROCK1 (Rho-kinase 1) may be the major Rho-kinase isoform that is opposed by ERK-MAPK signaling in IPC. These results indicate that ERK-MAPK signaling is required in IPC to oppose Rho-kinase activity in cardiomyocyte apoptosis in vivo.