A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jκ–dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease. (Cancer Res 2006; 66(3): 1517-25)