The role of glucocorticoids in the regulation of apoptosis remains incongruous. Here, we demonstrate that corticosterone protects neurons from apoptosis by a mechanism involving the cyclin-dependent kinase inhibitor p21^Waf1/Cip1. In primary cortical neurons, corticosterone leads to a dose- and Akt-kinase-dependent upregulation with enhanced phosphorylation and cytoplasmic appearance of p21^Waf1/Cip1 at Thr 145. Exposure of neurons to the neurotoxin ethylcholine aziridinium (AF64A) results in activation of caspase-3 and a dramatic loss of p21^Waf1/Cip1 preceding apoptosis in neurons. These effects of AF64A are reversed by pretreatment with corticosterone. Corticosterone-mediated upregulation of p21^Waf1/Cip1 and neuroprotection are completely abolished by glucocorticoid and mineralocorticoid receptor antagonists as well as inhibitors of PI3- and Akt-kinase. Both germline and somatically induced p21^Waf1/Cip1 deficiency abrogate the neuroprotection by corticosterone, whereas overexpression of p21^Waf1/Cip1 suffices to protect neurons from apoptosis. We identify p21^Waf1/Cip1 as a novel antiapoptotic factor for postmitotic neurons and implicate p21^Waf1/Cip1 as the molecular target of neuroprotection by high-dose glucocorticoids.