Abstract Calmodulin is a small (148 residues), ubiquitous, highly-conserved Ca 2+ binding protein serving as a modulator of many calcium-dependent processes. In this study, we followed, by means of molecular dynamics, the structural stability of the protein when one of its four bound Ca 2+ ions is removed, and compared it to a simulation of the fully Ca 2+ bound protein. We found that the removal of a single Ca 2+ ion from the N-lobe of the protein, which has a lower affinity for the ion, is sufficient to initiate a considerable structural rearrangement. Although the overall structure of the fully 4 Ca 2+ bound protein remained intact in the extended conformation, the Ca 2+-removed protein changed its conformation into a compact state. The observation that the 3 Ca 2+ loaded protein assumes a compacted solution state is in accord with experimental observation that the NSCP protein, which binds only three Ca 2+ ions, is natively in a compact state. Examination of the folding dynamics reveals a cooperation between the C-lobe, N-lobe, and the interdomain helix that enable the conformation change. The forces driving this conformational change are discussed.