The alterations of GO nanostructures after γ-ray irradiation in water, air and styrene with an absorbed dose of 200 kGy are systematically investigated. The interlayer structures of the ultimate products are confirmed to be remarkably different from each other due to the distinct changes of functional groups on single-sheets in various media. After irradiation in water, oxygen groups in graphite oxide are shown to be obviously decreased owing to the generation of reductive radicals by the decomposition of water molecules, which is reflected in the decrease of graphite oxide interlayer spacing. The interlayer distance of graphite oxide irradiated in air is found to be significantly increased, which may be attributed to the increase of the hydroxyl groups and the topological defects. However, the graphite oxide seems to be mainly exfoliated and functionalized by the intercalation of the monomers and the grafting of polystyrene chains when irradiated in styrene. It is expected that γ-ray irradiation in different media should be a promising strategy for manipulating nanostructures and properties of graphite oxide for improving its applicability in fields of composites, catalysts and sensors.