Aberrant activation of cell cycle proteins is believed to play a critical role in Alzheimer's disease (AD) pathogenesis; although, the molecular mechanisms leading to their activation in diseased neurons remain elusive. The goal of this study is to investigate the mechanistic link between Cdk5 deregulation and cell cycle re-activation in Aβ1-42-induced neurotoxicity. Using a chemical genetic approach, we identified Cdc25A, Cdc25B, and Cdc25C as direct Cdk5 substrates in mouse brain lysates. We show that deregulated Cdk5 directly phosphorylates Cdc25A, Cdc25B, and Cdc25C at multiple sites, which not only increases their phosphatase activities but also facilitates their release from 14-3-3 inhibitory binding. Cdc25A, Cdc25B, and Cdc25C in turn activate Cdk1, Cdk2 and Cdk4 kinases causing neuronal death. Selective inhibition of Cdk5 abrogates Cdc25 and Cdk activations in Aβ1-42-treated neurons. Similarly, phosphorylation-resistant mutants of Cdc25 at Cdk5 sites are defective in activating Cdk1, Cdk2, and Cdk4 in Aβ1-42-treated primary cortical neurons, underlining Cdk5-mediated Cdc25 activation as a major mechanism causing Cdks activation in AD pathogenesis. These results were further confirmed in human clinical samples which displayed higher Cdc25A, Cdc25B and Cdc25C activities which were coincident with increased Cdk5 activity in AD samples, as compared to age-matched controls. Inhibition of Cdk5 confers the highest neuroprotection against Aβ1-42 toxicity, while inhibition of Cdc25 was partially neuroprotective, further emphasizing a decisive role of Cdk5 deregulation in cell cycle-driven AD neuronal death.