Hyperphosphorylation of Tau protein and related neuron death is one of the most important characteristics of Alzheimer's disease. Our laboratory has shown that Tau protein is able to bind and protect DNA. It is still unknown whether phosphorylation affects the binding of Tau to DNA. Therefore, it is of importance to investigate the effect of phosphorylation on the interaction of Tau with DNA in cells. In this work, we treated N2a cells with formaldehyde and found that Tau protein was hyperphosphorylated in the cells under the experimental conditions. Phosphorylation was remarkably observed at both T181 and S396 of Tau protein in the cells in the presence of formaldehyde compared with those in the absence of formaldehyde. Cytoimmunofluorescence hardly showed that most of the nuclear phosphorylated Tau protein was co-localized with DNA, while Tau protein was partially co-localized with DNA in the absence of formaldehyde as control. Electrophoretic mobility shift assay (EMSA) showed that phosphorylated Tau catalyzed by GSK-3β reduced the interaction between Tau protein and DNA in vitro. These findings reveal that hyperphosphorylation declines Tau protein to protect DNA, and may thereafter lead to damage of DNA and even cell death, giving a novel viewpoint to the pathology of Alzheimer's disease.