The activity of Cdk5 and its regulatory subunit p35 is thought to be important in both normal brain function and neurodegenerative disease pathogenesis. Increased Cdk5 activity, via proteolytic cleavage of p35 to a p25 fragment by the calcium-activated protease calpain or by phosphorylation at Cdk5(Tyr15), can contribute to neurotoxicity. Nonetheless, our knowledge of regulation of Cdk5 activity in disease states is still emerging. Here we demonstrate that Cdk5 is activated by S-nitrosylation or reaction of nitric oxide (NO)-related species with the thiol groups of cysteine residues 83 and 157, to form SNO-Cdk5. We then show that S-nitrosylation of Cdk5 contributes to amyloid-β (Aβ) peptide-induced dendritic spine loss. Furthermore, we observed significant levels of SNO-Cdk5 in postmortem Alzheimer's disease (AD) but not in normal human brains. These findings suggest that S-nitrosylation of Cdk5 is an aberrant regulatory mechanism of enzyme activity that may contribute to the pathogenesis of AD.