The MgtE family of Mg(2+) transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full-length MgtE and of its cytosolic domain in the presence and absence of Mg(2+) suggested a Mg(2+)-homeostasis mechanism, in which the MgtE cytosolic domain acts as a 'Mg(2+) sensor' to regulate the gating of the ion-conducting pore in response to the intracellular Mg(2+) concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full-length structure precluded an unambiguous characterization of these regulatory divalent-cation-binding sites. Here, we showed that MgtE is a highly Mg(2+)-selective channel gated by Mg(2+) and elucidated the Mg(2+)-dependent gating mechanism of MgtE, using X-ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg(2+) homeostasis through cooperative Mg(2+) binding to the MgtE cytosolic domain.