The function of sodium iodide symporter (NIS) in mammary gland epithelial cells is essential for the accumulation of iodide in mother’s milk, which is the first source of iodide for the synthesis of thyroid hormones in the newborn. In addition to the lactating mammary gland, NIS expression has been also detected in breast tumors. Several hormones and ligands have been implicated in the functional expression of NIS in the mammary gland and breast cancer cell line models but the molecular determinants governing this expression are not yet identified. In this study we aimed to identify cis- and trans-acting elements regulating NIS expression in the breast cancer cell line MCF-7 in response to all-trans-retinoic acid (tRA), and to assess the possible role of 17-β-estradiol (E2) in regulating the expression of NIS. Using comparative bioinformatics, we have identified several regions that were conserved in human, mouse and rat in the sequences flanking and including the NIS gene. By using luciferase reporter assays, we have established that conserved clusters 3 and 4 respond to tRA in MCF-7. We have also shown that putative retinoic acid response elements controlling tRA-induced NIS expression in MCF-7 are located in the first intron of this gene. This tRA-responsive NIS expression was also correlated with the estrogen receptor status of mammary gland cell lines and we investigated roles of ERα in the regulation of NIS expression. We showed that the suppression of endogenous ERα by RNA interference resulted in down-regulation of both basal and tRA-induced NIS expression in MCF-7, furthermore, we have also shown that (E2) is capable of up-regulating NIS expression in MCF-7. In the ERα negative cell line MDA-MB-231, re-introduction of ERα resulted in NIS expression in a ligand independent manner. The role of ERα in the regulation of NIS expression was supported by the identification of an estrogen response element (ERE) in the promoter of NIS, this ERE was conserved in human, mouse and rat. We have also showed that this ERE could respond to E2 stimulation, and that ERα occupies the NIS promoter by binding to this novel element in vivo. These results indicate that E2 and ERα contribute to the regulation of NIS in the breast cancer cell line MCF-7.