BKca channels play a key role in the increased uterine artery blood flow in pregnancy, which is attenuated by chronic hypoxia during gestation. We examined transcriptional mechanisms of BKca β1 subunit promoter in ovine uterine arteries. We cloned a 2‐kb promoter of β1 gene in sheep, which contains several transcription factor binding sites with CpG dinucleotides in their core binding sequences, including ERα/SP1, SP1, and AP1. ERα/SP1, but not SP1 or AP1, sites were hypomethylated in uterine arteries of pregnant as compared with nonpregnant animals, which was reversed by chronic hypoxia. The binding of ERα and SP1 to the site was confirmed by EMSA with super shift and ERα and SP1 bind simultaneously to the site. CpG methylation inhibited the binding. Deletion of the ERα/SP1 binding site significantly attenuated reporter gene activity of β1 promoter. ChIP assay showed an increase in ERα and SP1 binding to β1 promoter in pregnant uterine arteries, which was abrogated by chronic hypoxia. In accordance, β1 protein expression was increased in pregnant uterine arteries, which was blocked by hypoxia. The results demonstrate a novel mechanism of promoter methylation in transcriptional regulation of BKca β1 subunit gene expression patterns in uterine arteries in response to pregnancy and hypoxia. (Supported in part by NIH grants HL89012 and HL110125).