In smooth muscle, the large-conductance calcium and voltage sensitive potassium channel (maxi-K) generate isoforms with disparate responses to contractile stimuli. We have previously shown that the human myometrium expresses high levels of the maxi-K channel isoform mK44. The studies presented here demonstrate that nardilysin convertase, a zinc-dependent metalloprotease of the insulinase family, regulates the plasma membrane expression of mK44 and its response to increases in intracellular calcium. We show that nardilysin convertase isoform 1 is present in human myometrium and co-localizes with mK44. Studies indicate that nardilysin convertase regulates: 1) retention of the mK44 C-terminal fragment in the endoplasmic reticulum in quiescent myometrial smooth muscle; and 2) calcium-induced translocation of mK44 to the plasma membrane. In mouse fibroblasts, nardilysin convertase significantly attenuates mK44-dependent current. In human myometrial smooth muscle cells, its inhibition promotes membrane localization of mK44 and an increase in maxi-K current. Overall, our data indicate that in human myometrium, nardilysin convertase and mK44 channels are a part of the molecular mechanism that regulates the excitability of smooth muscle cells.