Calcineurin (CaN) is a calcium/calmodulin-dependent protein phosphatase that has an important role in ischemia-induced apoptosis. The serine/threonine kinase, Akt, which is also known as protein kinase B, has an important role in the cell death/survival pathways. Akt is activated by its phosphorylation, which is positively regulated by phosphatidylinositol 3-kinase (PI3K) and negatively regulated by a class of protein phosphatases (PPs) in tissue. However, the relationship between CaN and Akt after transient ischemia remains unclear. In the present study, we investigated whether CaN is involved in neuronal cell apoptosis and Akt dephosphorylation that occur during ischemic injury. We examined the interdependence between CaN and Akt/protein kinase B (PKB) in the rat retina after transient ischemia. After ischemic damage, we detected changes in levels of CaN, Akt and Bad in rats in the presence or absence FK506, CaN inhibitor. Our results show that CaN cleavage reduced Akt phosphorylation at Thr308 and Ser473, and led to apoptosis via dephosphorylation of the proapoptotic Bcl-2 family member Bad. After treatment with FK506, Akt and Bad dephosphorylation was greatly reduced. The total number of TUNEL-positive neurons was reduced by intravitreal injection of FK506 after transient ischemia. These results indicate that CaN cleavage negatively regulates Akt phosphorylation and is involved in retinal cell apoptosis after transient ischemia.