Potassium (K) channels exhibit exquisite selectivity for conduction of K + ions over other cations, particularly Na + . High-resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K + ions, but not Na + ions, are perfectly coordinated. Using single-molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentration. The constrained conformation, essential for selective K + permeability, is stabilized by K + but not Na + ions. Mutations that render channels nonselective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild-type KirBac1.1 channels are K + selective in physiological conditions, Na + permeates in the absence of K + . Moreover, whereas K + gradients preferentially support 86 Rb + fluxes, Na + gradients preferentially support 22 Na + fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.