The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most differentiated thyroid tumours exhibit reduced iodide uptake, however, and the mechanisms accounting for this remain poorly understood. Pituitary tumor transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We recently reported that PTTG and its binding factor PBF, also a potent transforming gene which is over-expressed in thyroid cancer, repress expression of sodium iodide symporter (NIS) mRNA, and inhibit iodide uptake. We have now elucidated a mechanism by which PBF directly modulates NIS activity in vitro. In COS-7 and rat thyroid FRTL-5 subcellular localisation studies, PBF was directed to the membrane, and subsequently internalised into intracellular vesicles, co-localising with the tetraspanin CD63. Transient PBF over-expression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, and co-localisation with PBF and CD63. In contrast, localisation of the unrelated plasma membrane protein CD8 was unaffected by PBF transfection. Cell surface biotinylation assays in COS-7 cells confirmed a reduction in cell surface expression of NIS following PBF transfection compared with vector-only (VO) treatment (32±9% reduction, P=0.018, N=3). PTTG failed to influence NIS cell surface expression. In subsequent in vitro pull-down experiments, NIS and PBF proteins bound specifically to each other. To assess the functional consequence of this interaction, iodide uptake studies were performed in FRTL5 cells. PBF repressed iodide uptake compared to VO (39.4±5% reduction, P<0.001, N=10), whereas 3 mutants of PBF, lacking amino acids 28–93, 93–149 or 149–179 lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF, which is overexpressed in thyroid tumours, binds the sodium iodide symporter and alters its subcellular localisation, regulating its ability to uptake iodide.