Interaction of water and water-based solvents with graphene oxide (GO) has attracted much attention, due to the ability of GO to serve as a highly effective water filtration membrane. In this work, we study the evolution of the structure of GO in a partially reduced form, before and after being hydrated in high humidity conditions. X-ray diffraction (XRD) studies reveal that progressive thermal reduction leads to the increase in the microstructural disorder in the stacking of GO flakes. However, upon hydration of partially reduced GO, microstructural ordering is revealed. This ordered state is characterized by two XRD peaks with substantially smaller full-width-at-half-maximum (FWHM), when compared to the pre-hydration state. The peak corresponding to the sp 3 regions has larger d-spacing of ∼9.7 Å and an FWHM ∼6 times smaller compared to pre-hydration state, while the other peak corresponds to the ordered sp 2 regions with a d-spacing of ∼3.3 Å, observed at the characteristic graphitic peak position. Gravimetry studies on suspended films reveal both accelerated and diminished water permeation rates upon annealing when compared to unreduced GO films, which can be attributed to void-assisted permeation in the microstructurally disordered films. The hydrated films in a similar way show a permeation behavior that involves either the increase or decrease in water permeation rates in comparison with pre-hydrated samples. We reconcile to the gravimetry outcomes by suggesting the possibilities of both super-permeating channels and void assisted permeation, and the contribution of each of the mechanisms to the permeation flux. © 2018 Author(s).