Glycogen synthase kinase 3β (GSK3β) is an essential protein kinase that regulates numerous functions within the cell. One critically important substrate of GSK3β is the microtubule-associated protein tau. Phosphorylation of tau by GSK3β decreases tau–microtubule interactions. In addition to phosphorylating tau, GSK3β is a downstream regulator of the wnt signaling pathway, which maintains the levels of β-catenin. Axin plays a central role in regulating β-catenin levels by bringing together GSK3β and β-catenin and facilitating the phosphorylation of β-catenin, targeting it for ubiquitination and degradation by the proteasome. Although axin clearly facilitates the phosphorylation of β-catenin, its effects on the phosphorylation of other GSK3β substrates are unclear. Therefore in this study the effects of axin on GSK3β-mediated tau phosphorylation were examined. The results clearly demonstrate that axin is a negative regulator of tau phosphorylation by GSK3β. This negative regulation of GSK3β-mediated tau phosphorylation is due to the fact that axin efficiently binds GSK3β but not tau and thus sequesters GSK3β away from tau, as an axin mutant that does not bind GSK3β did not inhibit tau phosphorylation by GSK3β. This is the first demonstration that axin negatively affects the phosphorylation of a GSK3β substrate, and provides a novel mechanism by which tau phosphorylation and function can be regulated within the cell.