AbstractMitochondrial RNA splicing 2 (Mrs2), a eukaryotic CorA ortholog, enables Mg²⁺ to permeate the inner mitochondrial membrane and plays an important role in mitochondrial metabolic function. However, the mechanism by which Mrs2 permeates Mg²⁺ remains unclear. Here, we report four cryo-electron microscopy (cryo-EM) reconstructions of Homo sapiens Mrs2 (hMrs2) under various conditions. All of these hMrs2 structures form symmetrical pentamers with very similar pentamer and protomer conformations. A special structural feature of Cl⁻-bound R-ring, which consists of five Arg332 residues, was found in the hMrs2 structure. Molecular dynamics simulations and mitochondrial Mg²⁺ uptake assays show that the R-ring may function as a charge repulsion barrier, and Cl⁻ may function as a ferry to jointly gate Mg²⁺ permeation in hMrs2. In addition, the membrane potential is likely to be the driving force for Mg²⁺ permeation. Our results provide insights into the channel assembly and Mg²⁺ permeation of hMrs2.