Cation diffusion facilitator (CDF) proteins are a conserved family of transmembrane transporters that ensure cellular homeostasis of divalent transition metal cations. Metal cations bind to CDF protein's cytoplasmic C‐terminal domain (CTD), leading to closure from its apo open V‐shaped dimer to a tighter packed structure, followed by a conformational change of the transmembrane domain, thus enabling transport of the metal cation. By implementing a comprehensive range of biochemical and biophysical methods, we studied the molecular mechanism of metal binding to the magnetotactic bacterial CDF protein MamM CTD. Our results reveal that the CTD is rather dynamic in its apo form, and that two dependent metal‐binding sites, a single central binding site and two symmetrical, peripheral sites, are available for metal binding. However, only cation binding to the peripheral sites leads to conformational changes that lock the protein in a compact state. Thus, this work reveals how metal binding is regulating the sequential uptakes of metal cations by MamM, and extends our understanding of the complex regulation mechanism of CDF proteins. Database Structural data are available in RCSB Protein Data Bank under the accession numbers: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G64, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G55, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G5E and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G6I (for CS, C267S, CS‐C267S and W247A, respectively).