Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activation in vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg). Transgenic TTBK1 is highly expressed in subiculum and cortical pyramidal layers, and induces phosphorylated neurofilament aggregation. TTBK1-Tg mice show significant age-dependent memory impairment as determined by radial arm water maze test, which is associated with enhancement of tau and neurofilament phosphorylation, increased levels of p25 and p35, both activators of cyclin-dependent protein kinase 5 (CDK5), enhanced calpain I activity, and reduced levels of hippocampal NMDA receptor types 2B (NR2B) and D. Enhanced CDK5/p35 complex formation is strongly correlated with dissociation of F-actin from p35, suggesting the inhibitory mechanism of CDK5/p35 complex formation by F-actin. Expression of recombinant TTBK1 in primary mouse cortical neurons significantly downregulated NR2B in a CDK5- and calpain-dependent manner. These data suggest that TTBK1 in AD brain may be one of the underlying mechanisms inducing CDK5 and calpain activation, NR2B downregulation, and subsequent memory dysfunction.