Objective:To determine whether and how glycogen synthase kinase-3β (GSK-3β), an important component of phosphoinositide 3-kinases (PI3K) signaling pathway, regulates the methylation of protein phosphotase 2A (PP2A). Methods:By using molecular biological and pharmological approaches to alter the activity of PI3K signaling pathyway and its key molecule, GSK-3β, we investigated the regulation of PI3K pathway and GSK-3β on PP2A methylation by using Western blot analysis in cultured HepG2 and HEK-293T cells,By using GST-pull down, we determined the interaction between LCMT-1 and GSK-3. Results:We found that inhibition of PI3K pathway by PI3K inhibitor LY294002 increased the methylation level of PP2A catalytic subunit. In addition, inhibition of GSK-3β in cells leaded to the less methylation of PP2A catalytic subunit. We found that GST-GSK-3β could pull-down LCMT-1. Conclusion:PI3K signaling pathway is involved in the regulation of PP2A catalytic subunit methylation. Inhibition of GSK-3β suppresses the methylation. GSK-3β has a mutual effect with LCMT-1 and may regulate the methylation of PP2A through LCMT-1.