Understanding the regulation of myometrial smooth muscle excitability is important for developing an effective treatment for preterm labor. Large-conductance Ca²⁺– and voltage-sensitive potassium channels (maxi-K) promote uterine quiescence by generating repolarizing current. Our laboratory identified an alternatively spliced maxi-K channel isoform (mK44) that contains a 44 amino acid (aa) insertion in the first intracellular loop and is expressed predominantly in myometrial and aortic smooth muscle. Endogenous mK44 channels are localized in the endoplasmic reticulum (ER) in human myometrial smooth muscle cells (hMSMCs). This finding indicated that mK44 current does not contribute to hMSMC K⁺ current in the quiescent state. We hypothesized that mK44 channels express functional current on the cell membrane when induced by contractile stimuli. In hMSMCs after incubation with 20 mM caffeine, mK44 translocates to the cell membrane and induces hMSMC repolarization. To understand channel trafficking, mK44 was fused with a c-myc tag at the N-terminus (myc/mK44) and transiently expressed in hMSMCs. The N- and C-termini were found to have disparate localization with the C-termini localized in the ER, and the N-termini localized to the cell membrane. Upon incubation with caffeine, the C-termini translocated to the cell membrane and co-localized with the N-termini. Sequence analysis of the mK44 specific insert suggested that mK44 has a putative recognition motif (-RK-, aa 62-63) for the M16 family of peptidases and may undergo an endoproteolytic digest. An N-terminal fragment of ~10 kDa, corresponding to the predicted N-terminal peptide that would result from endoproteolytic cleavage of mK44, was detected in hMSMCs lysates. Mutating this motif generated a protein (R62A/V5) that localized to the ER in hMSMCs, like wild-type mK44, but failed to translocate to the cell membrane in response to caffeine. Heterologous expression of the N-termini (aa 1-62) and C-termini (aa 69-1157) of mK44 reconstituted a functional channel sensitive to IbTX on the cell membrane in HEK293F cells. The myc/mK44 channels did not undergo endoproteolytic digest when expressed in HEK293F cells or mouse fibroblasts suggesting a posttranslational modification specific for a myometrial smooth muscle. Thus, we have identified a novel mechanism to regulate hMSMCs excitability in response to prolonged exposure to certain contractile agents. This mechanism includes a specialized pool of K⁺ channels (mK44) that are normally retained in the ER and translocate to the cell membrane in response to sustained depolarization to generate repolarizing current and maintain hMSMC quiescence.