Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from all sections of the nephron and represent a reservoir for biomarker discovery. Exosomes can ‘shuttle’ protein, messenger RNA and microRNA between cells and may represent a new physiological signalling mechanism. To understand the mechanisms of exosome mediated signalling along the nephron we explored the uptake of exosomes by collecting duct cells. These cells were used because in vivo they are downstream in terms of urine flow along the nephron and are therefore exposed to exosomes from a range of upstream nephron segments. To explore exosome uptake, exosomes were fluorescently labelled then co-cultured with murine cortical collecting duct (mCCD) cells. In a subset of studies the mCCD cells were grown on transwell membranes to allow specific stimulation of apical or basolateral cellular compartments. To quantify exosome transfer the supernatant exosome concentration was measured by nanoparticle tracking analysis and the intracellular exosome concentration was measured by flow cytometry. Confocal laser scanning microscopy was used for intra-cellular visualisation and localisation of labelled exosomes. Labelled exosomes were internalised by mCCD cells and this uptake was significantly increased following stimulation with the vasopressin analogue desmopressin (dDAVP) (percentage of cells containing labelled exosomes: dDAVP 38% ± 8.4% vs control 16.7% ± 4.8%, p<0.001). Tolvaptan, a selective vasopressin V2 receptor antagonist, decreased exosome uptake following dDAVP stimulation (tolvaptan and dDAVP 15.6% ± 3.65% vs dDAVP 38.03% ±8.39%, p<0.001). Endothelin-1, a peptide with a known inhibitory effect on vasopressin action, and dynamin inhibition decreased exosome uptake following dDAVP stimulation. Using mCCD cell monolayers, the site of dDAVP action was confirmed to be basolateral, but exosome uptake was from both apical and basolateral compartments. In conclusion, this study is the first to demonstrate that vasopressin regulates exosome uptake in the kidney, in a V2 receptor mediated process through clathrin-dependent endocytosis. This understanding will allow us to better define the role of exosomes in kidney physiology and develop exosomes as therapeutic agents for kidney disease.