Radioiodine ablation of differentiated thyroid cancers and their metastases utilises the ability of the thyroid to accumulate iodide. However, most differentiated thyroid tumours exhibit reduced iodide uptake, with the mechanisms underlying this remaining poorly understood. Pituitary tumor transforming gene (PTTG) binding factor (PBF) is a proto-oncogene over-expressed in thyroid tumours, which we have shown to repress sodium iodide symporter (NIS) mRNA expression and inhibit iodide uptake. We now describe a mechanism by which PBF directly modulates NIS activity in vitro. In COS-7 and rat thyroid FRTL-5 subcellular localisation studies, PBF and NIS demonstrated co-localisation within intracellular vesicles. A physical interaction between PBF and NIS proteins was identified in GST pull-down and co-immunoprecipitation experiments. Transient PBF over-expression was associated with an increase in NIS staining within intracellular vesicles. Cell surface biotinylation assays in COS-7 cells confirmed a reduction in cell surface expression of exogenous NIS following PBF transfection compared with vector-only (VO) treatment (32±9% reduction, P=0.018, N=3), whilst total NIS protein levels remained constant. PBF and NIS both co-localised with the tetraspanin CD63, an endosomal marker associated with clathrin-dependent endocytosis but not caveolin-1, a marker for caveolin-dependent lipid trafficking endocytosis, suggesting these proteins are internalised via clathrin-dependent endocytosis. To assess the functional consequence of the NIS-PBF interaction, iodide uptake studies were performed in FRTL-5 cells. PBF repressed iodide uptake compared to VO (39.4±5% reduction, P<0.001, N=10). In contrast, three deletion mutants of PBF, which do not co-localise with either NIS or CD63 within intracellular vesicles, were unable to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds the sodium iodide symporter and alters its subcellular localisation, regulating its ability to uptake iodide. Given that PBF is over-expressed in thyroid tumours, these findings have profound implications for ablative radioiodine treatment.