O-GlcNAcylation of proteins is a recently discovered post-translational modification of nuclear and cytoplasmic proteins. This modification is similar to protein phosphorylation rather than to classical protein glycosylation of membrane and secreted proteins. Both O-GlcNAcylation and phosphorylation modify the hydroxyl group of serine or threonine residues of tau, the effect of O-GlcNAcylation on phosphorylation of tau was studied. The level of O-GlcNAcylation in differenciated PC12 cells was modulated by changing the concentration of the donor of O-GlcNAcylation and activities of the key enzymes, then, the consequent changes of tau phosphorylation at various phosphorylation sites were examined by using Western blot developed with phosphorylation-dependent and site-specific tau antibodies. It was found that O-GlcNAcylation modulated phosphorylation of tau at many phosphorylation sites and in a site-specific manner. Increased protein O-GlcNAcylation induced a decrease in tau phosphorylation at most of phosphorylation sites, and vise versa. These results suggest that O-GlcNAcylation negatively modulates tau phosphorylation at most phosphorylation sites. Therefore, these studies provide novel insight into the regulation of tau phosphorylation and the molecular mechenism of abnormal hyperphosphorylation of tau in Alzheimer disease brain.