Expression of the Keratin 13 (KRT13) gene, which encodes a cytoskeletal protein thought to play important roles in breast cancer growth and metastasis, is differentially regulated by estradiol (E2) and the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene. While stimulation of KRT13 by tamoxifen is robust and prolonged, stimulation by E2 is more transient and raloxifene has virtually no effect. To investigate the mechanistic basis for the differential ligand regulation of KRT13, we have defined the regulatory regions of KRT13, compared gene expression by E2 and SERMs, and explored the magnitudes and time courses of estrogen receptor (ER) and cofactor recruitment patterns on these regions. Using a ChIP scanning approach and reporter transactivation assays, we identified a 2.5 kb upstream ER-binding regulatory region for KRT13. Directed composite mutations in this region revealed that three estrogen response elements and three Sp1 sites were involved in its ligand-dependent regulation. Differential recruitment of ERalpha and cofactors to the KRT13 regulatory sites paralleled the different time course and magnitude of regulation by these ligands: there was almost no ERalpha or cofactor recruitment with raloxifene, whereas there was strong, prolonged ER recruitment and histone acetylation with tamoxifen, and an early and more transient recruitment with E2. Taken together, our results suggest that the different ligand regulations of KRT13 are due to ligand-differential recruitment of ER and coactivators, and they provide insight into the mechanisms responsible for the different agonistic activities and differential gene regulation by estradiol and the SERMs tamoxifen and raloxifene.