The fast C-type inactivation displayed by the voltage-activated potassium channel hERG plays a critical role in the repolarization of cardiac cells, and malfunction caused by nonspecific binding of drugs or naturally occurring missense mutations affecting inactivation can lead to pathologies. Because of its impact on human health, understanding the molecular mechanism of C-type inactivation in hERG represents an advance of paramount importance. Here, long-time scale molecular dynamics simulations, free energy landscape calculations, and electrophysiological experiments are combined to address the structural and functional impacts of several disease-associated mutations. Results suggest that C-type inactivation in hERG is associated with an asymmetrical constricted-like conformation of the selectivity filter, identifying F627 side-chain rotation and the hydrogen bond between Y616 and N629 as key determinants. Comparison of hERG with other K+ channels suggests that C-type inactivation depends on the degree of opening of the intracellular gate via the filter-gate allosteric coupling.