Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value. Here, we show that heparin interacts with uptake of tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy (TEM), with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using binding assays and microscopy. We found that the block in EVs uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin as a therapeutic for disease in which EVs play a role.