Amyloid precursor proteins (APPs) are processed by β-, γ-, and ε-secretases and caspase-3 to generate C-terminal fragments of APP (APP-CTFs), which may contribute to the pathology of Alzheimer's disease (AD). In addition to amyloid plaques and neurofibrillary tangles, AD brains contain Hirano bodies, which are rod-like structures mostly composed of actin and the actin-binding protein, cofilin. However, the mechanisms underlying the formation of cofilin-actin rods are still unknown. In this study, we aim to elucidate the effects of APP-CTFs on the actin-depolymerizing factor [(ADF)/cofilin]. Our data indicate that transfection with APP-CT99 and APP-CT57 may increase the phosphorylation level of Ser3 of ADF/cofilin and Thr508 of LIM-kinase 1 in rat primary cortical neuronal cultures. S3 peptide, a synthetic peptide competitor of LIM-kinase 1 for ADF/cofilin phosphorylation and an inhibitor of APP-CTFs, induced ADF/cofilin phosphorylation. In comparison with the wild-type mouse, the APP-CT transgenic mouse showed increased immunoreactivity of phosphorylated cofilin (p-cofilin) in the brain. Treatment with DAPT, an inhibitor of γ-secretase, resulted in a decrease in p-cofilin protein level in the group transfected with full-length APP-695. Transfection with the mutant APP-CTF with a deleted YENPTY domain resulted in no significant increase in p-cofilin level. Thus, APP-CTFs induced cofilin phosphorylation to facilitate nuclear translocation. These results suggest a relationship between APP-CTFs and ADF/cofilin that may be suggestive of a new toxic pathway in the pathology of AD.