The CorA/Mrs2 family of pentameric proteins are cardinal for the influx of Mg2+ across cellular membranes, importing the cation to mitochondria in eukaryotes. Yet, the conducting and regulation mechanisms of permeation remain elusive, particularly for the eukaryotic Mrs2 members. Here, we report closed and open Mrs2 cryo-electron microscopy structures, accompanied by functional characterization. Mg2+ flux is permitted by a narrow pore, gated by methionine and arginine residues in the closed state. Transition between the conformations is orchestrated by two pairs of conserved sensor-serving Mg2+-binding sites in the mitochondrial matrix lumen, located in between monomers. At lower levels of Mg2+, these ions are stripped, permitting an alternative, symmetrical shape, maintained by the RDLR motif that replaces one of the sensor site pairs in the open conformation. Thus, our findings collectively establish the molecular basis for selective Mg2+ influx of Mrs2 and an auto-ligand-gating regulation mechanism. Here, the authors demonstrate how Mrs2, critical for import of Mg2+ into the mitochondria, transitions from open and closed five-fold symmetric states to control the influx of Mg2+ using an auto-ligand-gating regulation mechanism.