BK channel is ubiquitously expressed. In vascular cells it is expressed with the accessory β1 subunit, where they play an important role in the modulation of arterial tone and blood pressure. Modifications on the cholesterol concentration in the cellular membrane can lead to changes on the BK channel activity, which could affect the interaction of BK channel with other regulatory molecules like 17β-Estradiol (E2). The aim of this research was to evaluate the effect of the changes on cholesterol membrane concentration in the Estradiol-BK channel interaction. HEK 293 cells were transfected with human BKα (U11058) and/or BKβ1 (U25138) harbored in pcDNA3.1 plasmids. Cholesterol depletion was conducted in transfected cells with MbCD treatment. BK membrane expression was analyzed by flow cytometry and confocal microscopy. Cells were treated with E2-BSA-FITC and binding assays were carried out using confocal microscopy and flow cytometry. Patch clamp recordings in inside out configuration was used to determinate the E2 modulatory effect on BK channel. Cholesterol depletion increased BKα subunit expression in α/β1 co-transfection (from 85±2.3 to 94±1.2%) with any change in the β1 subunit expression alone (from 89±1.5 to 93±1.2) or in co-expression with α (from 92±1.3 to 95±1.0). These results suggest that the BK channel expression is modulated by membrane cholesterol concentration. After cholesterol depletion we found an increase in the E2 binding to the β1 subunit, when it is expressed alone (from 5.5±0.2 to 6.8±0.3. n = 3; P ≤ 0.01) but not in α/β1 co-transfection. When we evaluated the effect of cholesterol depletion on the E2-BK channel interaction, we observed that the activation kinetics of the BKα/β1 complexes was not affected. However, cholesterol depletion prevents the modulatory effect of E2 on the channel, since the half-maximal activation potential (V0.5) remains the same after the E2 stimuli (from 137±4mV to 140±4mV after depletion). These data suggest that changes in membrane cholesterol concentration causes the loss of the modulatory effect of the E2 on the BKα/β1 channels, which is not related with the BKα or β1 subunits expression.