The retinoblastoma (Rb) suppressor associated protein 46 (RbAp46) is a subunit of chromatin modifying and remodeling complexes. Previously, we found that RbAp46 functions as a potent growth inhibitor. It is also a downstream effector of the Wilms' tumor suppressor, WT1. The findings that expression levels of WT1 were down-regulated in breast cancer cell lines and in subsets of primary breast tumors led us to investigate the possible role of RbAp46 in breast cancer tumorigenesis. Here, we found that RbAp46 expression levels were decreased in five established breast cancer cell lines compared to a normal mammary gland epithelial cell line. To investigate the effect of constitutive expression of RbAp46 on the transformed phenotypes of breast cancer cells, we established stable cell lines that constitutively express exogenous RbAp46 using three breast cancer cell lines, MCF-7, MDA-MB-231 and MDA-MB-436. We have found that RbAp46 expression suppressed colony formation of these breast cancer cells in soft-agar, and inhibited tumor formation of these cells in nude mice. Our data demonstrated that constitutive RbAp46 expression suppresses the transformed phenotypes of breast cancer cells, and suggested that dysregulation of RbAp46 expression may contribute to breast cancer tumorigenesis.