AbstractThe structure of calbindin D9k with two substitutions was determined by X‐ray crystallography at 1.8‐Å resolution. Unlike wild‐type calbindin D9k, which is a monomeric protein with two EF‐hands, the structure of the mutated calbindin D9k reveals an intertwined dimer. In the dimer, two EF‐hands of the monomers have exchanged places, and thus a 3D domain‐swapped dimer has been formed. EF‐hand I of molecule A is packed toward EF‐hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF‐hands, promotes the conversion of monomers to 3D domain‐swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D9k. Once formed, the calbindin D9k dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D9k dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.