Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca²⁺, and the binding of Ca²⁺ is required for the activation of calmodulin. Once bound to Ca²⁺, calmodulin acts as part of a calcium signal transduction pathway by modifying its interactions with various target proteins such as kinases or phosphatases.

Calmodulin is a small, highly conserved protein that is 148 amino acids long (16.7 kDa). The protein has two approximately symmetrical globular domains (the N- and C- domains) each containing a pair of EF hand motifs separated by a flexible linker region for a total of four Ca²⁺ binding sites, two in each globular domain. In the Ca²⁺-free state, the helices that form the four EF-hands are collapsed in a compact orientation, and the central linker is disordered; in the Ca²⁺-saturated state, the EF-hand helices adopt an open orientation roughly perpendicular to one another, and the central linker forms an extended alpha-helix in the crystal structure, but remains largely disordered in solution. The C-domain has a higher binding affinity for Ca²⁺ than the N-domain.

Calmodulin is structurally quite similar to troponin C, another Ca²⁺-binding protein containing four EF-hand motifs. However, troponin C contains an additional alpha-helix at its N-terminus, and is constitutively bound to its target, troponin I. It therefore does not exhibit the same diversity of target recognition as does calmodulin.

Calmodulin's ability to recognize a tremendous range of target proteins is due in large part to its structural flexibility. In addition to the flexibility of the central linker domain, the N- and C-domains undergo open-closed conformational cycling in the Ca²⁺-bound state. Calmodulin also exhibits great structural variability, and undergoes considerable conformational fluctuations, when bound to targets. Moreover, the predominantly hydrophobic nature of binding between calmodulin and most of its targets allows for recognition of a broad range of target protein sequences. Together, these features allow calmodulin to recognize some 300 target proteins exhibiting a variety of CaM-binding sequence motifs.

Binding of Ca²⁺ by the EF-hands causes an opening of the N- and C-domains, which exposes hydrophobic target-binding surfaces. These surfaces interact with complementary nonpolar segments on target proteins, typically consisting of groups of bulky hydrophobic amino acids separated by 10–16 polar and/or basic amino acids. The flexible central domain of calmodulin allows the protein to wrap around its target, although alternate modes of binding are known. "Canonical" targets of calmodulin, such as myosin light-chain kinases and CaMKII, bind only to the Ca²⁺-bound protein, whereas some proteins, such as NaV channels and IQ-motif proteins, also bind to calmodulin in the absence of Ca²⁺. Binding of calmodulin induces conformational rearrangements in the target protein via "mutually induced fit", leading to changes in the target protein's function.

Calcium binding by calmodulin exhibits considerable cooperativity, making calmodulin an unusual example of a monomeric (single-chain) cooperative binding protein. Furthermore, target binding alters the binding affinity of calmodulin toward Ca²⁺ ions, which allows for complex allosteric interplay between Ca²⁺ and target binding interactions. This influence of target binding on Ca²⁺ affinity is believed to allow for Ca²⁺ activation of proteins that are constitutively bound to calmodulin, such as small-conductance Ca²⁺-activated potassium (SK) channels.

Although calmodulin principally operates as a Ca²⁺ binding protein, it also coordinates other metal ions. For example, in the presence of typical intracellular concentrations of Mg²⁺ (0.5–1.0 mM) and resting concentrations of Ca²⁺ (100 nM), calmodulin's Ca²⁺ binding sites are at least partially saturated by Mg²⁺. This Mg²⁺ is displaced by the higher concentrations of Ca²⁺ generated by signaling events. Similarly, Ca²⁺ may itself be displaced by other metal ions, such as the trivalent lanthanides, that associate with calmodulin's binding pockets even more strongly than Ca²⁺. Though such ions distort calmodulin's structure and are generally not physiologically relevant due to their scarcity in vivo, they have nonetheless seen wide scientific use as reporters of calmodulin structure and function.

Calmodulin mediates many crucial processes such as inflammation, metabolism, apoptosis, smooth muscle contraction, intracellular movement, short-term and long-term memory, and the immune response. Calcium participates in an intracellular signaling system by acting as a diffusible second messenger to the initial stimuli. It does this by binding various targets in the cell including a large number of enzymes, ion channels, aquaporins and other proteins. Calmodulin is expressed in many cell types and can have different subcellular locations, including the cytoplasm, within organelles, or associated with the plasma or organelle membranes, but it is always found intracellularly. Many of the proteins that calmodulin binds are unable to bind calcium themselves, and use calmodulin as a calcium sensor and signal transducer. Calmodulin can also make use of the calcium stores in the endoplasmic reticulum, and the sarcoplasmic reticulum. Calmodulin can undergo post-translational modifications, such as phosphorylation, acetylation, methylation and proteolytic cleavage, each of which has potential to modulate its actions.