Mitochondrial RNA splicing protein 2 (MRS2) forms a major magnesium (Mg 2+ ) entry channel into the matrix. While MRS2 contains two transmembrane domains that constitute a pore, most of the protein resides within the matrix. Yet, the precise structural and functional role of this obtrusive amino terminal domain (NTD) in human MRS2 function is unknown. Here, we show that the MRS2 NTD self-associates into a homodimer, contrasting the pentameric assembly of CorA, an orthologous bacterial channel. Mg 2+ and calcium suppress lower and higher order oligomerization of MRS2 NTD, while cobalt has no effect on the NTD but disassembles full-length MRS2. Mutating pinpointed residues mediating Mg 2+ binding to the NTD, not only selectively decreases Mg 2+ binding affinity ∼7-fold but also abrogates Mg 2+ binding-induced changes in secondary, tertiary and quaternary structures. Disruption of NTD Mg 2+ binding strikingly potentiates mitochondrial Mg 2+ uptake in wild-type and Mrs2 knockout cells. Our work exposes a mechanism for human MRS2 autoregulation by negative feedback from the NTD and identifies a novel gain of function mutant with broad applicability to future Mg 2+ signaling research.