Graphene oxide (GO) sheets offer multiple propositions as a vector system, including high loading capacity due to its large surface-to-volume ratio, versatile functionalization, high dispersibility and relative low cytotoxicity. Here, we have investigated the interactions of GO flakes with in vitro (U-87 MG three-dimensional spheroids) and in vivo (U-87 MG orthotopic xenograft) models of glioblastoma. In vitro experiments with U-87 MG spheroids, devoid of stromal or immune compartments, showed that GO flakes passively translocated deeply into the spheroids with little internalization in tumor cells, did not cause cytotoxicity, and only induced small changes in gene and protein expression that did not exacerbate tumor cell proliferation. In vivo , intracranially administered GO also showed extensive distribution throughout the tumor mass and had no impact on tumor growth and progression for the duration of the study. Interestingly, GO internalization within tumor cells in vivo was similarly scarce as in the in vitro experiments, with the majority of the sheets preferentially taken up by IBA1 + microglia/macrophages. Overall, our results indicated that GO sheets could offer deep and homogenous distribution throughout the tumor mass and could be used as vectors to target the macrophage glioblastoma compartment.