Abstract TLE1 is a Groucho-related transcriptional repressor protein that exerts survival and antiapoptotic function in several cellular systems and has been implicated in the pathogenesis of cancer. In the present study, we found that TLE1 is a regulator of anoikis in normal mammary epithelial and breast carcinoma cells. The induction of apoptosis following loss of cell attachment to the extracellular matrix (anoikis) in untransformed mammary epithelial MCF10A cells was associated with significant downregulation of TLE1 expression. Forced expression of exogenous TLE1 in these cells promoted resistance to anoikis. In breast cancer cells, TLE1 expression was significantly upregulated following detachment from the extracellular matrix. Genetic manipulation of TLE1 expression via overexpression and downregulation approaches indicated that TLE1 promotes the anoikis resistance and anchorage-independent growth of breast carcinoma cells. Mechanistically, we show that TLE1 inhibits the Bit1 anoikis pathway by reducing the formation of the proapoptotic Bit1-AES complex in part through sequestration of AES in the nucleus. The mitochondrial release of Bit1 during anoikis as well as exogenous expression of the cytoplasmic localized Bit1 or its cell death domain induced cytoplasmic translocation and degradation of nuclear TLE1 protein. These findings indicate a novel role for TLE1 in the maintenance of anoikis resistance in breast cancer cells. This conclusion is supported by an immunohistochemical analysis of a breast cancer tissue array illustrating that TLE1 is selectively upregulated in invasive breast tumors relative to noninvasive ductal carcinoma in situ and normal mammary epithelial tissues. Mol Cancer Res; 10(11); 1482–95. ©2012 AACR.