Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has demonstrated that the regulatory domain of CaN (RD) is disordered. The binding of CaM to CaN results in RD folding. Folding of the RD in turn causes the autoinhibitory domain (AID) located C-terminal to the RD to be ejected from CaN’s active site. This binding-induced disorder-to-order transition is responsible for the activation of CaN by CaM. In this work, we explore the nature of the disorder in the RD and its transition to an ordered state, demonstrating that the RD exists in a compact disordered state that undergoes further compaction upon CaM binding. We also demonstrate that a single CaM molecule is responsible for binding to and activating CaN. Finally, we determine that the CaM binding to CaN induces an amphipathic helix (the distal helix) C-terminal to the CaM binding region. The distal helix undergoes a hairpin-like chain reversal in order to interact with the surface of CaM, resulting in the removal of the AID from CaN’s active site. We employ site-directed mutagenesis, size-exclusion chromatography, protein crystallography, circular dichroism spectroscopy, fluorescence anisotropy and correlation spectroscopy, and phosphatase activity assays to investigate the ordering of CaN’s regulatory domain, the stoichiometry of CaN:CaM binding, and the impact of the distal helix on CaM activation of CaN.