A novel separation system consisting of thermosensitive membranes was proposed for macromolecular separation, and the system was evaluated in this study. The pore surface of a porous substrate was covered with a thermosensitive N-isopropylacrylamide (NIPAM) grafted polymer, which enabled the hydrophobicity of the pore surface to: be varied drastically by a slight temperature change. A solution containing hydrophobic and hydrophilic solutes was continuously supplied to the feed side, and the membrane temperature was changed stepwise between above and below the lower critical solution temperature (LCST) of NIPAM. Above the LCST, the hydrophobic solutes adsorb onto the hydrophobic;pore surfaces; hence, only the hydrophilic solutes can pass through the membrane. Lowering the temperature below the LCST caused the pore surface to become hydrophilic and the adsorbed solutes to; desorb from the pore surface. In addition, the hydrophobic solutes condensed in the permeate side; Nonionic surfactant NP-10 was used as a model solute for the proposed system, and the validity of the proposed system was demonstrated using the membrane prepared.