Many Ser/Thr protein kinases, to be fully activated, are obligated to introduce a phospho-Ser/Thr in their activation loop. Presently, the similarity of activation loop between two crystal complexes, i.e. glycogen synthase kinase 3beta (GSK3beta)-AMPNP and GSK3beta-sulfate ion complex, indicates that the activation segment of GSK3beta is preformed requiring neither a phosphorylation event nor conformational changes. GSK3beta, when participated in glycogen synthesis and Wnt signaling pathways, possesses a unique feature with the preference of such substrate with a priming phosphate. Experimental mutagenesis proved that the residue arginine at amino acid 96 mutations to lysine (R96K) or alanine (R96A) selectively abolish activity on the substrates involved in glycogen synthesis signaling pathway. Based on two solved crystal structures, wild type (WT) and two mutants (R96K and R96A) GSK3beta-ATP-phospho-Serine (pSer) complexes were modeled. Molecular dynamics simulations and energy analysis were employed to investigate the effect of pSer involvement on the GSK3beta structure in WT, and the mechanisms of GSK3beta deactivation due to R96K and R96A mutations. The results indicate that the introduction of pSer to WT GSK3beta generates a slight lobe closure on GSK3beta without any remarkable changes, which may illuminate the experimental conclusion, whereas the conformations of GSK3beta and ATP undergo significant changes in two mutants. As to GSK3beta, the affected positions distribute over activation loop, alpha-helix, and glycine-rich loop. Based on coupling among the mentioned positions, the allosteric mechanisms for distorted ATP were proposed. Energy decomposition on the residues of activation loop identified the important residues Arg96 and Arg180 in anchoring the phosphate group.