Layer-stacked graphene oxide (GO) membranes, in which unique two-dimensional (2D) water channels are formed between two neighboring GO nanosheets, have demonstrated great potential for aqueous phase separation. Subjects of crucial importance are to fundamentally understand the interlayer spacing (i.e., channel height) of GO membranes in aqueous environment, elucidate the mechanisms for water transport within such 2D channels, and precisely controlling the interlayer spacing to tune the membrane separation capability for targeted applications. In this investigation, we used an integrated quartz crystal mass balance (QCM-D) and ellipsometry to experimentally monitor the interlayer spacing of GO, reduced GO and crosslinked GO, respectively, in aqueous solution, and found that crosslinking can effectively prevent GO from swelling and precisely control the interlayer spacing. We then used molecular dynamics simulation to study the mass transport inside the 2D channels, and prove that the chemical functional groups on GO plane dramatically slow down water transport in the channels. Our findings on GO structure and water transport provide a necessary basis for further tailoring and optimizing the design and fabrication of GO membranes in various separation applications.