Hexagonal mesoporous silica membranes were prepared by the sol-gel route using a silicon alkoxide as silica precursor and alkyltrimethylammonium bromides to form the templating liquid crystal mesophase. These 2D mesoporous structures consist of cylindrical pores whose axes are preferentially aligned parallel to the surface of the substrate due to interfacial interactions at both air-layer and layer-substrate interfaces. In order to increase the pore accessibility and to improve the permeability of the hexagonal porous layers, the starting solutions were seeded with silica nanoparticles. This seeding promotes a heterogeneous nucleation of the templating mesophase by creating additional interfaces inside the hybrid gelling solution. X-ray diffraction techniques and transmission electron microscopy demonstrated clearly that the preferential ordering of the cylindrical pores parallel to the surface can be destroyed by nanoparticle seeding. Nitrogen permeation measurements were carried out on unseeded and seeded hexagonal silica membranes. As expected from the previous static characterizations, the membrane permeability is strongly increased ( x 10) in the case of seeded layers. The measured permeabilities were discussed from the expected model of gas transport in mesoporous materials.