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Mg2+ translocation across cellular membranes is crucial for a myriad of physiological processes. Eukaryotic Mrs2 transporters are distantly related to the major bacterial Mg2+ transporter CorA, the structure of which displays a bundle of giant [alpha]-helices forming a long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. Here, a functional and structural analysis of the regulatory domain of the eukaryotic Mg2+ channel Mrs2 from the yeast inner mitochondrial membrane is presented using crystallography, genetics, biochemistry and fluorescence spectroscopy. Surprisingly, the fold of the Mrs2 regulatory domain bears notable differences compared with the related bacterial channel CorA. Nevertheless, structural analysis showed that analogous residues form functionally critical sites, notably the hydrophobic gate and the Mg2+-sensing site. Validation of candidate residues was performed by functional studies of mutants in isolated yeast mitochondria. Measurements of the Mg2+ influx into mitochondria confirmed the involvement of Met309 as the major gating residue in Mrs2, corresponding to Met291 in CorA.