This study presents the fabrication of a biomimetic reverse osmosis (RO) membrane from a supported lipid bilayer (SLB) and gramicidin A (GA) onto polymeric substrates via electrostatic interaction and by applying hydraulic pressure. GA, a cation-selective ionophore, has high permeability for water molecules and monovalent cations in a lipid bilayer, and was therefore used as a water channel molecule. Cationic liposomes containing GA were electrostatically adsorbed onto an anionic polymeric nanofiltration membrane. The adsorbed liposomes were ruptured by applying hydraulic pressure and transformed into flat lipid bilayers. SLB formation was confirmed by salt rejection measurement and fluorescence recovery after photobleaching. The defect-free SLB was formed on the polymeric nanofiltration membrane by applying over 0.15 MPa pressure. Phosphorus quantification showed that the formed SLB was not composed of a single lipid bilayer but approximately six lipid bilayers. GA incorporation into the SLB and control of the GA conformation on the SLB effectively improved water permeability. Moreover, the GA-incorporated SLB showed high NaCl rejection (>97%). Monovalent cations, Na+ ions, were rejected to maintain electric neutrality. The formed SLB showed hydraulic pressure resistance and high salt rejection at adequate levels for RO membranes. (C) 2015 Elsevier B.V. All rights reserved.