Long-QT syndrome (LQTS) is a life-threatening cardiac arrythmia whereby ventricular recovery time is extended, prolonging the QT interval as seen on an electrocardiogram. Affecting 1 in 2000 people, LQTS predisposes sufferers to an increased risk of suffering catastrophic cardiac events, typically resulting in sudden cardiac death (SCD). The most common cause of LQTS are congenital mutations in the Kv7.1 voltage-gated potassium channel which facilitates one of the main ventricular repolarising currents (IKs). Calmodulin (CaM) is a ubiquitous calcium-sensing protein which regulates Kv7.1 activity. Several mutations in CaM have been identified in human patients displaying LQTS phenotypes, suggesting a key role in the molecular aetiology of LQTS. However, the molecular mechanisms of CaM-associated LQTS remain unclear. Here, we present novel data regarding the biophysical properties of four LQTS-associated mutations located at calcium co-ordinating residues within the C-lobe of CaM, and their interactions with a CaM-binding domain (Helix B) at the C -terminus of the alpha subunit (KCNQ1) of the Kv7.1 channel. Using circular dichroism, we showed that single amino acid substitutions confer significant changes to the thermostability and secondary structure of CaM variants. Additionally, using equilibrium calcium binding titrations, we showed that the affinity of CaM C-lobe for calcium can be reduced up to 2-fold. Isothermal titration calorimetry experiments provided evidence of both calcium-dependent and independent binding of CaM to Helix B (Kv7.1). In its calcium-free state, CaM binds Helix B through hydrophobic interactions with moderate affinity (Kd = 2.2 ± 0.2 μM). Ca2+ dependent binding to Helix B is considerably higher affinity, with over a 2000-fold reduction in dissociation constant (Kd). LQTS-associated CaM variants were found to reduce affinity for Helix B with the largest reductions found in fourth EF hand mutants. These mutants also adopted a highly consistent, alternative complex with Helix B compared to WT CaM as determined through HSQC 1H-15N NMR. Using whole cell configuration voltage-clamp electrophysiology, we demonstrated that CaM modulates the Kv7.1 channel to produce larger currents without altering channel activation kinetics. The IKs current was found to be Ca2+ sensitive, in response to increases in cytosolic Ca2+, larger IKs currents are generated (from 47 ± 6 to 150 ± 31 pA pF-1 at +60 mV, n = 22 and 9 respectively). Interestingly, LQTS-associated CaM mutants impair this Ca2+ sensitivity by reducing current density. Here we outline mechanistic insights as to how LQTS-associated CaM variants contribute to electrical disease of the heart, mutations in the highly conserved structure of CaM appear to perturb protein structure, its ability to bind calcium, and disrupt complex formation with the KCNQ1 channel. This results in reduced generation of the IKs current, ultimately decreasing the repolarising capacity of cells and therefore extending the QT interval.