Absorption of dietary iodide, presumably in the small intestine, is the first step in iodide (I^-) utilization. From the bloodstream, I^- is actively taken up via the Na⁺/I^- symporter (NIS) in the thyroid for thyroid hormone biosynthesis and in such other tissues as lactating breast, which supplies I^- to the newborn in the milk. The molecular basis for intestinal I^- absorption is unknown. We sought to determine whether I^- is actively accumulated by enterocytes and, if so, whether this process is mediated by NIS and regulated by I^- itself. NIS expression was localized exclusively at the brush border of rat and mouse enterocytes. In vivo intestine-to-blood transport of pertechnetate, a NIS substrate, was sensitive to the NIS inhibitor perchlorate. Brush border membrane vesicles accumulated I^- in a sodium-dependent, perchlorate-sensitive manner with kinetic parameters similar to those of thyroid cells. NIS was expressed in intestinal epithelial cell line 6 (IEC-6), and I^- uptake in these cells was also kinetically similar to that in thyrocytes. I^- downregulated NIS protein expression and its own NIS-mediated transport both in vitro and in vivo. We conclude that NIS is functionally expressed on the apical surface of enterocytes, where it mediates active I^- accumulation. Therefore, NIS is a significant and possibly central component of the I^- absorption system in the small intestine, a system of key importance for thyroid hormone biosynthesis and thus systemic intermediary metabolism.