CorA is the major transport system responsible for Mg(2+) uptake in bacteria and can functionally substitute for its homologue Mrs2p in the yeast inner mitochondrial membrane. Although several CorA crystal structures are available, the molecular mechanism of Mg(2+) uptake remains to be established. Here we use electron paramagnetic resonance spectroscopy, electrophysiology and molecular dynamic simulations to show that CorA is regulated by cytoplasmic Mg(2+) acting as a ligand and elucidate the basic conformational rearrangements responsible for Mg(2+)-dependent gating. Mg(2+) unbinding at the divalent cation sensor triggers a conformational change that leads to the inward motion of the stalk helix, which propagates to the pore-forming transmembrane helix TM1. Helical tilting and rotation in TM1 generates an iris-like motion that increases the diameter of the permeation pathway, triggering ion conduction. This work establishes the molecular basis of a Mg(2+)-driven negative feedback loop in CorA as the key physiological event controlling Mg(2+) uptake and homeostasis in prokaryotes.