Ablative therapy for thyroid cancer depends upon the adequate uptake of radioiodine via the sodium iodide symporter (NIS). However, thyroid tumours frequently show reduced iodide uptake, impacting on prognosis. We previously demonstrated that the proto-oncogene PBF binds NIS and modulates its subcellular localisation. We now demonstrate that PBF binds the thyroid hormone transporter MCT8 in GST-pulldown assays, and alters MCT8 subcellular localisation in vitro. To investigate PBF function further we carried out immunofluorescent studies in COS-7 cells. PBF was localised predominantly within intracellular vesicles, whereas a PBF deletion mutant lacking the C-terminal 30 amino acids was targeted to the plasma membrane. We hypothesised therefore that PBF shuttles in and out of the plasma membrane, and that loss of a tyrosine-based internalisation signal (YXXΦ motif) within the deleted region caused the accumulation of PBF within the plasma membrane. Discrete mutation of the critical tyrosine residue (Y174) and the hydrophobic residue (F177) resulted in increased plasma membrane staining, confirming the YXXΦ motif as an active internalisation signal. Further, a functional signal peptide and transmembrane domain were identified when deletion of residues 3–32 and 94–120 respectively resulted in protein retention in the endoplasmic reticulum. PBF has two putative N-linked glycosylation sites at amino acids 45 and 54. Following Western analysis of COS-7 cell lysates, multiple PBF bands between 25 and 37 kDa were all reduced to ~20 kDa through the use of glycosylation inhibitors and glycosylation mutants. Interestingly, a mutant lacking residues 39–93 showed an increase in membrane staining, suggesting that glycosylation is not essential for membrane targeting, but is required for efficient internalisation. These data further characterise the transmembrane glycoprotein PBF, which given that it regulates the subcellular expression of NIS, may provide a therapeutic target in the enhancement of radioiodine therapy in relatively radioresistant thyroid cancers and their metastases.