Adenocarcinomas of the prostate arise as multifocal heterogeneous lesions as the likely result of genetic and epigenetic alterations and deranged cell-cell communication. Notch signaling is an important form of intercellular communication with a role in growth/differentiation control and tumorigenesis. Contrasting reports exist in the literature on the role of this pathway in prostate cancer (PCa) development. We show here that i) compared to normal prostate tissue, Notch1 expression is significantly reduced in a substantial fraction of human PCas while it is unaffected or even increased in others; ii) acute Notch activation both inhibits and induces process networks associated with prostatic neoplasms; iii) down-modulation of Notch1 expression and activity in immortalized normal prostate epithelial cells increases their proliferation potential, while increased Notch1 activity in PCa cells suppresses growth and tumorigenicity through a Smad3-dependent mechanism involving p21WAF1/CIP1; iv) prostate cancer cells resistant to Notch growth inhibitory effects retain Notch1-induced upregulation of pro-oncogenic genes, like EPAS1 and CXCL6, also overexpressed in human PCas with high Notch1 levels. Taken together, these results reconcile conflicting data on the role of Notch1 in prostate cancer.