Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads and in neurofibrillary tangle containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomic analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared to undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of alpha-Tubulin, alpha-Actinin-4, Spinophilin and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin and alpha-Tubulin. A neo-epitope antiserum to alpha-Tubulin cleaved by Caspase-6 immunostains neurons, neurofibrillary tangles, neuropil threads and neuritic plaques in Alzheimer disease and co-localizes with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in AD could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in absence of cell death. This study provides novel insights into the patho-physiology of Alzheimer disease.