A nanometer-sized signal-responsive gate was fabricated by self-assembly of ionizable polypeptide brushes on a gold-coated porous membrane, and its responsiveness in water was visualized by atomic force microscopy. The gating was regulated by the conformational change of poly(glutamic acid) that undergoes a helix-coil transition in response to pH. As a result, water permeation through the membrane was reversibly regulated by pH. The permeability could be adjusted by varying the length of the polypeptide brush. This type of membrane will be useful for the precise control of substance permeation.