Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in the United States. Early diagnosis is rare and treatment options are limited. The Notch pathway has recently emerged as a candidate drug target. Notch signaling regulates the proliferation of pancreatic progenitors during embryogenesis and is reactivated in the earliest PDAC precursor lesions known as PanINs. This provides a rationale for targeting Notch signaling in these tumors using Gamma-Secretase Inhibitors (GSIs), which block Notch receptor cleavage and activation. Aims: Here, we investigated the therapeutic effect of GSI (MRK-003) in vivo using a genetically engineered mouse models and in cultured murine and human PDAC cell lines. Methods: A series of murine and human PDAC cell lines were treated with GSI and assessed for proliferation and growth in soft agar. In addition, mice engineered to sustain Cre-mediated activation of Kras(G12D) and heterozygous inactivation of p53 in the pancreas were randomized and either treated with vehicle (n=21) or with GSI (n=12). In both groups treatment were perfomed for a period of 4–7 weeks. The effect of GSI on tumor latency and Notch pathway was evaluated. Results: GSI caused a significant decrease in proliferation and soft agar colony formation at concentrations as low as 1.5µM in cell lines were tested. Growth inhibition was associated with a dose-dependent inhibition of the Notch target gene Hes1 in both human and murine cells. The in vivo data revealed the Notch inhibition caused a delay in tumor development and a suppression of premalignant pancreatic lesions. Conclusion: Our data support the potential oncogenic role of Notch in the development of PDAC and suggest that down-regulation of Notch signaling by GSI may be an effective strategy for prevention and/or treatment of PDAC.