Two-dimensional (2D) lamellar membranes are highly advantageous in molecular separations. However, the permeance-rejection trade-off is always a major challenge, since the permeant transport mostly occurs in single-spacing channels with undesired microenvironments. Inspired by the structure of aquaporins, we design alternating dual-spacing channel graphene oxide (GO) membranes, with locally tailored chemical microenvironments, that give high permeance, high rejection and high stability in organic solvent nanofiltration. This unique structure is easily constructed by in situ intercalating and cross-linking scattered sub-5 nm silica nanoparticles in the GO interlayers. The hydrophilic nanoparticles locally widen the interlayer channels to enhance the solvent permeance. In the alternating nanoparticle-free areas, the GO layers simultaneously bend and the π–π interactions retain the narrow and hydrophobic channels, promoting high solute rejection. With a 10-fold increase in water permeance and unaffected rejection, the dual-spacing channel membranes exhibit one of the best performances for organic solvent nanofiltration. The methanol permeance reaches 290 L m−2 h−1 bar−1, with more than 90% rejection of dyes larger than 1.5 nm. This new approach of designing hierarchical channels in 2D materials can be used for a wide spectrum of applications.