Calmodulin (CaM), a Ca2+-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca2+-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report thatCaMbinds to two isoforms of the hKv7.4 channel in a Ca2+- independent manner but that only the long isoform (hKv7.4a) is regulated by Ca2+/CaM. Ca2+/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca2+/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca2+on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca2+/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.