Dietary I(-) absorption in the gastrointestinal tract is the first step in I(-) metabolism. Given that I(-) is an essential constituent of the thyroid hormones, its concentrating mechanism is of significant physiological importance. We recently described the expression of the Na(+)/I(-) symporter (NIS) on the apical surface of the intestinal epithelium as a central component of the I(-) absorption system and reported reduced intestinal NIS expression in response to an I(-)-rich diet in vivo. Here, we evaluated the mechanism involved in the regulation of NIS expression by I(-) itself in enterocytes. Excess I(-) reduced NIS-mediated I(-) uptake in IEC-6 cells in a dose- and time-dependent fashion, which was correlated with a reduction of NIS expression at the plasma membrane. Perchlorate, a competitive inhibitor of NIS, prevented these effects, indicating that an increase in intracellular I(-) regulates NIS. Iodide induced rapid intracellular recruitment of plasma membrane NIS molecules and NIS protein degradation. Lower NIS mRNA levels were detected in response to I(-) treatment, although no transcriptional effect was observed. Interestingly, I(-) decreased NIS mRNA stability, affecting NIS translation. Heterologous green fluorescent protein-based reporter constructs revealed a significant repressive effect of the I(-)-targeting NIS mRNA 3 untranslated region. In conclusion, excess I(-) downregulates NIS expression in enterocytes by virtue of a complex mechanism. Our data suggest that I(-) regulates intestinal NIS mRNA expression at the post-transcriptional level as part of an autoregulatory effect of I(-) on its own metabolism.