RKE-1 cells induced to overexpress activated Notch1 (RKE-ER-N^ic) exhibit increased cyclin D1 transcripts and become transformed. However, the oncogenic pathway of Notch1-induced transformation is not known. Here, we use mutational analysis to functionally identify the sole region of the cyclin D1 promoter that responds to activated Notch1. The same region responds to activated Notch4 as well as to physiologic Notch ligand-induced Notch receptor signaling. The cyclin D1 gene was subsequently found to be a physiologic target of Notch signaling in Pofut1^−/− mouse embryos defective in canonical Notch signaling and in embryos with an inactivating mutation in Notch1. To determine if Notch1-induced cyclin D1 expression in RKE-ER-N^ic cells plays a direct role in transformation, cyclin D1 up-regulation was inhibited using a cyclin D1 antisense cDNA. We report here that transformation of RKE-ER-N^ic cells is dependent on increased expression of cyclin D1 protein, which represents a new mechanism of Notch1-induced transformation. (Cancer Res 2006; 66(15): 7562-70)