The p53 tumor suppressor protein is a key regulator of cellular functions including responses to numerous stress signals, and triggers apoptosis in many cell types, including neurons. The major mechanisms known to regulate p53 stabilization and activation include phosphorylation and ubiquitin ligase-mediated proteasomal degradation. Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is most active in the central nervous system and plays a variety of roles in neuronal degeneration. Here, we demonstrate for the first time that Cdk5 interacts with p53 and increases its stability through posttranslational regulation, leading to accumulation of p53, particularly in the nucleus. We show that Cdk5 phosphorylates p53 on Ser15, Ser33 and Ser46 in vitro, and that increased Cdk5 activity in the nucleus mediates these phosphorylation events in response to genotoxic and oxidative stresses. Cdk5 mediates disruption of the interaction between p53 and Hdm2 (also known as Mdm2), and prevents Hdm2-induced p53 ubiquitylation and downregulation. Cdk5 additionally enhances phosphorylation-dependent binding of the p300 coactivator, inducing acetylation of p53. Cdk5-stabilized p53 protein is transcriptionally active, resulting in the induction of pro-apoptotic genes and subsequent mitochondria-mediated apoptosis in response to genotoxic or oxidative stress. Collectively, these novel findings help define the mechanisms underlying neuronal apoptosis occurring as a result of Cdk5-mediated p53 stabilization and transcriptional activation.