The Na(+)/I(-) symporter (NIS (SLC5A5)) is a transmembrane glycoprotein that mediates active iodide uptake into thyroid follicular cells. NIS-mediated iodide uptake in thyroid cells is the basis for targeted radionuclide imaging and treatment of differentiated thyroid carcinomas and their metastases. Furthermore, NIS is expressed in many human breast tumors but not in normal non-lactating breast tissue, suggesting that NIS-mediated radionuclide uptake may also allow the imaging and targeted therapy of breast cancer. However, functional cell surface NIS expression is often low in breast cancer, making it important to uncover signaling pathways that modulate NIS expression at multiple levels, from gene transcription to posttranslational processing and cell surface trafficking. In this study, we investigated NIS regulation in breast cancer by MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling, an important cell signaling pathway involved in oncogenic transformation. We found that MEK inhibition decreased NIS protein levels in all-trans retinoic acid/hydrocortisone-treated MCF-7 cells as well as human breast cancer cells expressing exogenous NIS. The decrease in NIS protein levels by MEK inhibition was not accompanied by a decrease in NIS mRNA or a decrease in NIS mRNA export from the nucleus to the cytoplasm. NIS protein degradation upon MEK inhibition was prevented by lysosome inhibitors but not by proteasome inhibitors. Interestingly, NIS protein level was correlated with MEK/ERK activation in human breast tumors from a tissue microarray. Taken together, MEK activation appears to play an important role in maintaining NIS protein stability in human breast cancers.