Recent studies of ERs in breast cancer have demonstrated the existence of ERbeta in addition to ERalpha. Some clinical data indicated that ERbeta had prognostic value for patient's survival, which suggested that ERbeta plays a key role in breast cancer development and metastasis. To test this hypothesis, we generated an ERbeta high-expression cell line by reintroduced human ERbeta cDNA into MDA-MB-435 cells. We demonstrated that ERbeta exerted multiple tumor-stimulative effects on human breast carcinoma cells both in vivo and in vitro. In in vitro studies, ERbeta was able to increase the proliferation and invasion of MDA-MB-435 cells significantly, while these effects were totally estradiol independent. Also, this stimulation was characterized by downregulation of p21 and upregulation of MMP-9, as well as transcriptional factor Est-1. In in vivo studies, we also demonstrated that ERbeta-transfected MDA-MB-435 cells grew much faster and had more pulmonary metastasis than mock or wild-type cells in nude mice. In ERbeta-transfected MDA-MB-435 xenografts, ERbeta caused significant reduction in p21 protein levels. Similar effects of ERbeta on MMP-9 and Ets-1 expression noted in vitro studies were also observed in the in vivo studies. These in vitro and in vivo studies indicated that ERbeta exerted multiple stimulative effects on breast cancer development and metastasis.