Selective Estrogen Receptor Modulators (SERMs) are estrogen receptor (ER) ligands whose relative agonist/antagonist activities vary in a cell and promoter dependent manner. The molecular basis underlying this selectivity can be attributed to the ability of these ligands to induce distinct alterations in ER structure leading to differential recruitment of coactivators and corepressors. Whether SERM activity is restricted to synthetic ligands or if molecules exist in vivo that function in an analogous manner remains unsolved. However, the recent observation that oxysterols bind ER and antagonize the actions of 17-estradiol (E2) on the vascular wall suggests that this class of ligands possesses SERM activity. We demonstrate here that 27-hydroxycholesterol (27HC), the most prevalent oxysterol in circulation, functions as a SERM, the efficacy of which varies when assessed with different endpoints. Importantly, 27HC positively regulates both gene transcription and cell proliferation in cellular models of breast cancer. Using combinatorial peptide phage display we have determined that 27HC induces a unique conformational change in both ER and ER, distinguishing it from E2 and other SERMs. Thus, as with other ER ligands, it appears that the unique pharmacological activity of 27HC relates to its ability to impact ER structure and modulate cofactor recruitment. Cumulatively these data indicate that 27HC is an endogenous SERM with partial agonist activity in breast cancer cells and suggest that it may influence the pathology of breast cancer. Moreover, given the product-precursor relationship between 27HC and cholesterol, our findings have implications with respect to breast cancer risk in obese/hypercholesteremic individuals.