Glycogen synthase kinase 3β (GSK3β) is a ubiquitous serine/threonine kinase that plays a pivotal role in many biological processes. GSK3β catalyzes the transfer of γ-phosphate of ATP to the unique substrate Ser/Thr residues with the assistance of two natural activating cofactors Mg2+. Interestingly, the biological observation reveals that a non-native Ca2+ ion can inhibit the GSK3β catalytic activity. Here, the inhibitory mechanism of GSK3β by the displacement of native Mg2+ at site 1 by Ca2+ was investigated by means of 80 ns comparative molecular dynamics (MD) simulations of the GSK3β···Mg2+-2/ATP/ Mg2+-1 and GSK3β···Mg2+-2/ATP/Ca2+-1 systems. MD simulation results revealed that using the AMBER point charge model force field for Mg2+ was more appropriate in the reproduction of the active site architectural characteristics of GSK3β than using the magnesium-cationic dummy atom model force field. Compared with the native Mg2+ bound system, the misalignment of the critical triphosphate moiety of ATP, the erroneous coordination environments around the Mg2+ ion at site 2, and the rupture of the key hydrogen bond between the invariant Lys85 and the ATP Oβ2 atom in the Ca2+ substituted system were observed in the MD simulation due to the Ca2+ ion in active site in order to achieve its preferred sevenfold coordination geometry, which adequately abolish the enzymatic activity. The obtained results are valuable in understanding the possible mechanism by why Ca2+ inhibits the GSK3β activity and also provide insights into the mechanism of Ca2+ inhibition in other structurally related protein kinases. Proteins 2013. © 2012 Wiley Periodicals, Inc.