The Notch proteins constitute a family of transmembrane receptors that play a pivotal role in cellular differentiation, proliferation and apoptosis. Although it has been recognized that excess Notch signaling is potentially tumorigenic, little is known about precise mechanisms through which dysregulated Notch signaling induces neoplastic transformation. Here we demonstrate that Notch signaling has a transcriptional cross-talk with transforming growth factor-β (TGF-β) signaling, which is well characterized by its antiproliferative effects. TGF-β-mediated transcriptional responses are suppressed by constitutively active Notch1, and this inhibitory effect is canceled by introduction of transcriptional coactivator p300. We further show that this blockade of TGF-β signaling is executed by the sequestration of p300 from Smad3. Moreover, in a human cervical carcinoma cell line, CaSki, in which Notch1 is spontaneously activated, suppression of Notch1 expression with small interfering RNA significantly restores the responsiveness to TGF-β. Taken together, we propose that Notch oncoproteins promote cell growth and cancer development partly by suppressing the growth inhibitory effects of TGF-β through sequestrating p300 from Smad3. (Cancer Sci 2005; 96: 274 –283)