Ca²⁺/calmodulin (Ca²⁺/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca²⁺/CaM is not fully understood. Ca²⁺/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca²⁺/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca²⁺/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca²⁺/CaM with dissociation constants (K_d(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca²⁺-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca²⁺-dependent association at resting [Ca²⁺] of 50–100 nM is indicated in these complexes as one of the CaM Ca²⁺ binding sites displays high affinity with K_d of 70 and 30 nM for Ca²⁺, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca²⁺/CaM in a dose dependent manner, further cementing Ca²⁺/CaM as a regulator of gap junction function. The motion of a stretched CaM–CL2 complex compacting upon Ca²⁺ binding may bring about the Ca²⁺/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.