Notch signaling is a conserved signaling pathway that participates in many aspects of mammary gland development and homeostasis, and has extensively been associated with breast tumorigenesis. Here, to unravel the as yet debated role of Notch3 in breast cancer development, we investigated its expression in human breast cancer samples and effects of its loss in mice. Notch3 expression was very weak in breast cancer cells and was associated with good patient prognosis. Interestingly, its expression was very strong in stromal cells of these patients, though this had no prognostic value. Mechanistically, we demonstrated that Notch3 prevents tumor initiation via HeyL-mediated inhibition of Mybl2, an important regulator of cell cycle. In the mammary glands of Notch3-deficient mice, we observed accelerated tumor initiation and proliferation in a MMTV-Neu model. Notch3-null tumors were enriched in Mybl2 mRNA signature and protein expression. Hence, our study reinforces the anti-tumoral role of Notch3 in breast tumorigenesis.