Estrogens exert their biological action via both genomic and non-genomic mechanisms. Proteins different from classical estradiol receptors are believed to mediate the latter effects. Here we demonstrate that the maxi-K channel functions as an estrogen-binding protein in transfected HEK293 cells. Whole-cell maxi-K channel currents and protein expression were attenuated by exposure to either 17α- or 17β-estradiol. This effect was dose-dependent for 17β-estradiol at concentrations ranging from 10 nM to 1 μM, while 17α-estradiol inhibited channel expression only at 1 μM. These effects were mediated by direct low affinity binding of estradiol to the maxi-K channel but not to its accessory β1-subunit, as revealed by cell membrane estradiol binding assays. However, specific binding of estradiol to the channel was facilitated by the presence of the β1 subunit. Addition of MG-132, a blocker of proteasomal degradation, stabilized channel expression. These data suggest that channel down-regulation is mediated by estrogen-induced proteasomal degradation, similar to the pathway used for estrogen receptor degradation. Membrane expression of endogenous maxi-K channels in cultured vascular smooth muscle cells was also attenuated by prolonged exposure to 17α- and 17β-estradiol. Thus our studies demonstrate that estrogen binds to maxi-K channels and may directly regulate channel expression and function. These results will have important implications in understanding estradiol-induced effects in multiple tissues including vascular smooth muscle.