The estrogen receptor (ER) is a key regulator of proliferation and differentiation in breast cancer cells. In the present study, the effect of steroid and xenobiotic receptor (SXR) on 17β-estradiol (E2)-induced transcription through ERα was studied. SXR augmented ER-mediated transcription in the presence of E2 in MCF-7 breast cancer-derived cells and CV-1 fibroblast-derived cells. On the other hand, SXR alone did not affect the estrogen response element (ERE)-containing promoter activity in CV-1 cells. SXR did not directly bind to ERα or ERE in vitro, indicating that SXR may affect ER-mediated transcription by altering cofactor binding to ER. Although SXR did not alter the binding between ERα and p300/CBP interacting protein (p/CIP), it decreased the binding of a specific corepressor, silencing mediator of retinoid and thyroid hormone receptors (SMRT) to liganded ERα as assessed by mammalian two-hybrid, glutathione S-transferase pull-down, immunoprecipitation and newly developed Liquid Chemiluminescent DNA Pull-Down Assays. These results indicate that SXR augmented ER-mediated transcription by dissociating SMRT from ERα. Thus, the expression of SXR in breast cancer cells may alter the ER signaling, which may play crucial role for growth and differentiation of breast cancer cells.