The magnesium channel controls Mg2+ concentration in the cell and plays an indispensable role in biological functions. The crystal structure of the Magnesium Transport E channel suggested that Mg2+ hydrated by 6 water molecules is transported through a selection filter consisting of COO groups on two Asp residues. This Mg2+ motion implies successive pairing with OOC-R and dissociation mediated by water molecules. For another divalent ion, however, it is known that RCOO Ca2+ cannot be separated even with 12 water molecules. From this discrepancy, we probe the structure of Mg2+(CH3COO)(H2O)4–17 clusters by measuring the infrared spectra and monitoring the vibrational frequencies of COO with the help of quantum chemistry calculations. The hydration by (H2O)6 is not enough to induce ion separation, and partially-separated or separated pairs are formed from 10 water molecules at least. These results suggest that the ion separation between Mg2+ and carboxylate ions in the selection-filter of the MgtE channel not only results from water molecules in their first hydration shell, but also from additional factors including water molecules and protein groups in the second solvation shell of Mg2+.