Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERα and ERβ. ERα mediates the proliferative effect of estrogen in breast cancer cells, whereas ERβ seems to be antiproliferative. We engineered ERα-positive T47D breast cancer cells to express ERβ in a Tet-Off–regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERβ. The presence of ERβ resulted in a reduction in tumor growth. Comparison of the ERβ-expressing and non-ERβ–expressing tumors revealed that the expression of ERβ caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor β (PDGFβ). In cell culture, with the Tet-Off–regulated ERβ-expressing cells, expression of ERβ decreased expression of VEGF and PDGFβ mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFβ proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFβ promoter constructs revealed a repressive effect of ERβ at the promoter level of these genes. Taken together, these data show that introduction of ERβ into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)