A computational study was performed on the Mg2+-free conformations of the small guanine nucleotide-binding proteins (GNBPs): Ras, Rho, Rab, Arf, and Ran, which were complexed with GDP. Molecular dynamics (MD) simulation was executed for each complex for the duration of 3.0 ns to investigate the effects of Mg2+ ions on the GNBPs' structure. The results indicated that all Mg2+-free GNBPs formed a groove between the switch region and the nucleotide-binding site. In some GNBP families, the release of Mg2+ was reported to play an important role in binding the guanine nucleotide-exchanging factor (GEF) promoting the GDP/GTP exchange reaction. Interestingly, the grooves, which appeared in the MID simulations, were similar to the grooves experimentally observed in the GNBP-GEF complex. We also calculated the Mg2+-bound GNBPs to compare with the Mg2+-free forms. No groove was observed in the Mg2+-bound GNBPs. These results demonstrated a regulatory role of Mg2+ ion to prepare a template for the GEF binding. Moreover, the results suggested that the release of Mg2+ ion lead to the GEF-GNBP binding.