Magnesium ions (Mg2+) are crucial for various biological processes. A bacterial Mg2+ channel, MgtE, tightly regulates the intracellular Mg2+ concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane alpha helices forming a central pore, and intracellular soluble domains constituting a Mg2+ sensor. The ion selectivity for Mg2+ over Ca2+ resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg2+ and Ca2+. Using site-directed muta- genesis, vibrational bands at 1421 (Mg2+), 1407 (Mg2+), similar to 1440 (Ca2+), and 1390 (Ca2+) cm(-1) were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of the Mg2+-sensitive carboxylate bands, suggesting a highly optimized geometry for accommodating a Mg2+ ion. The dependency of the vibrational changes on Mg2+ and Ca2+ concentrations revealed the presence of a two different classes of binding sites: a high affinity site for Mg2+ (K-d approximate to 0.3 mM) with low Ca2+ affinity (K-d approximate to 80 mM), and a medium affinity site for Mg2+ (K-d approximate to 2 mM) and Ca2+ (K-d approximate to 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively. With the aid of molecular dynamics simulation and normalmode analysis by quantum chemistry, we confirm that changes in carboxylate bands of the high affinity binding site originate from Asp432 in the central cavity.