Nuclear factor kappa B (NF-κB) transcriptionally regulates several genes involved in initiating uterine contractions. A key factor controlling NF-κB activity is its translocation to the nucleus. In myometrial smooth muscle cells (MSMCs), this translocation can be stimulated by the inflammatory molecule lipopolysaccharide (LPS) or by blocking the potassium calcium-activated channel subfamily M alpha 1 (KCNMA1 or BK_Ca) with paxilline (PAX). Here, we sought to determine the mechanism by which blocking BK_Ca causes NF-κB-p65 translocation to the nucleus in MSMCs. We show that LPS- and PAX-induced NF-κB-p65 translocation are similar in that neither depends on several mitogen-activated protein kinase pathways, but both require increased intracellular calcium (Ca²⁺). However, the nuclear transport inhibitor wheat germ agglutinin prevented NF-κB-p65 nuclear translocation in response to LPS but not in response to PAX. Blocking BK_Ca located on the plasma membrane resulted in a transient NF-κB-p65 nuclear translocation that was not sufficient to induce expression of its transcriptional target, suggesting a role for intracellular BK_Ca. We report that BK_Ca also localizes to the nucleus and that blocking nuclear BK_Ca results in an increase in nuclear Ca²⁺ in MSMCs. Together, these data suggest that BK_Ca localized on the nuclear membrane plays a key role in regulating nuclear Ca²⁺ and NF-κB-p65 nuclear translocation in MSMCs. Summary Sentence Blocking of BK channels by paxilline in myometrial smooth muscle cells increases nuclear calcium levels and translocation of NF-κB-p65.