By exploiting the canonical function of the sodium iodide symporter (NIS), ablative radioiodide therapy is an effective treatment for thyroid cancer. However, a subset of patients are unable to accumulate sufficient radioiodide due to decreased expression and/or plasma membrane localisation of NIS. Radioiodide therapy has been proposed as a viable treatment for breast cancer, but is hampered by low levels of NIS membrane localisation. Currently, the regulation of NIS trafficking to the plasma membrane is ill-defined. Mass spectrometry was performed on proteins co-immunoprecipitating with lentivirally expressed NIS in whole cell and plasma membrane extracts. NIS function was assessed following knockdown, overexpression and pharmacological inhibition of shortlisted interactors using radioiodide uptake assays. Interactors were validated by co-immunoprecipitation and proximity ligation assays. NIS activity was significantly altered by ADP-ribosylation factor 4 (ARF4) and valosin containing protein (VCP) in TPC1 thyroid and MDA-MB-231 breast cancer cells lentivirally-expressing NIS. ARF4 downregulation decreased radioiodide uptake by 75 and 44%, and VCP downregulation increased radioiodide uptake by 71 and 56%, in thyroid and breast cells, respectively. Transient overexpression of these genes significantly reversed siRNA effects on NIS function. Co-immunoprecipitation assays confirmed NIS interacts with ARF4 and VCP in vitro, and proximity ligation assays revealed the subcellular sites of interaction. TCGA data analysis of 58 matched papillary thyroid cancers revealed ARF4 was significantly repressed and VCP highly upregulated in thyroid cancer, providing a putative explanation for repressed NIS function. Pharmacological inhibitor studies demonstrated Eeyarestatin-1 and NMS-873 could overcome VCP inhibition of NIS function, implicating the endoplasmic reticulum-associated degradation pathway as critical to NIS processing. Further, we identified that NIS is ubiquitylated in vitro, and suggest this as the possible mechanism through which VCP alters NIS function. These studies thus identify two new potential therapeutic targets for enhancing radioiodide uptake in patients with radioiodide-refractory thyroid cancer.