A mesoporous membrane composed of nanochannels with a uniform diameter has a potential use for precise size-exclusive separation of molecules. Here, we report a novel method to form a hybrid membrane composed of silica-surfactant nanocomposite and a porous alumina membrane, by which size-selective transport of molecules across the membrane becomes possible. The nanocomposite formed inside each columnar alumina pore was an assembly of surfactant-templated silica-nanochannels with a channel diameter of 3.4 nm; the channel direction being predominantly oriented along the wall of the columnar alumina pore. Molecules could be transported across the membrane including the silica-surfactant nanocomposite with a capability of nanometre-order size-exclusive separation. Our proposed membrane system has a potential use not only for separation science, but also catalysis and chip technologies.