The activity of protein phosphatase-2A (PP-2A) is significantly suppressed in the brain of Alzheimer's disease (AD) patients, but the mechanism is not understood. Here, we found an in vivo association of glycogen synthase kinase 3beta (GSK-3beta) with inhibitor-2 of PP-2A (I(2)(PP-2A)). The activation of GSK-3 resulted in accumulation of I(2)(PP-2A) with concomitant suppression of PP-2A activity and increases of tau phosphorylation in HEK293, N2a and PC12 cells, while inhibition of GSK-3 caused decreases of I(2)(PP-2A) with increased PP-2A activity and decreased tau phosphorylation. A positive correlation between GSK-3beta and I(2)(PP-2A) (R=0.9158) and a negative correlation between GSK-3beta and PP-2A (R=-0.9166) were detected. GSK-3 activation did not affect I(2)(PP-2A) mRNA level, while it increased the mRNA level of a heterogeneous ribonucleoprotein A18 (hnRNP A18). The activation of GSK-3 increased the expression and the activity of proteasome system. It suggests that activation of GSK-3 inhibits PP-2A through up-regulation of I(2)(PP-2A) with hnRNP A18-involved mechanism.