Graphene oxide (GO) nanosheets with atomic thickness and tunable physicochemical properties have been considered as promising nanobuilding blocks for fabrication of separation membranes with impressive performance. There are two kinds of molecular transport channels in laminar GO membranes, interlayer nanochannels formed by adjacent nanosheets and intrinsic defects/pores/edges of GO nanosheets. It has been demonstrated that precisely controlling the transport pathways at the angstrom level, through reduction, molecule/cation cross-linking, intercalation, physical confinement, electric field adjustment, pore creation, and defect sealing, can greatly improve the separation performance of GO membranes. Herein, we first briefly review the fabrication strategies of GO membranes and then comprehensively discuss the merits and mechanisms of controlling the transport pathways of GO membranes for liquid separation applications including static diffusion, pressure-driven filtration, and pervaporation.