Estrogen receptor (ERalpha) plays an important role in the development and progression of breast cancer. Several recent studies have demonstrated that expression of human X-box binding protein 1 (XBP-1) is associated with ERalpha status in breast tumors and overexpressed in a subset of breast tumors. XBP-1 has two splicing variants, which were designated as XBP-1S and XBP-1U, respectively. However, little is known about the expression pattern of XBP-1S and XBP-1U in breast cancer cells and about their roles in ERalpha signaling. In this study, the expression of two splicing forms of XBP-1 was detected in breast cancer cell lines with RT-PCR. Estrogen response element (ERE) -containing luciferase reporter assay was used to determine the effects of XBP-1S and XBP-1U on the transcription activity of ERalpha in MDA-MB-435 breast cancer cells. The result showed that both XBP-1S and XBP-1U enhanced the transcription activity of ERalpha in a hormone-independent and dose-dependent manner and the activity of of XBP-1S is higher than that of XBP-1U. Enhancement of ERE-containing luciferase reporter gene expression by XBP-1S and XBP-1U was dependent on ERalpha. These data suggest that XBP-1S and XBP-1U may play important roles in breast cancer growth and progression through ERalpha signaling.