Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of <5%. Genetic analysis of PDAC patient samples has shown that specific disease-associated mutations are correlated with histologically defined stages of neoplastic progression in the ductal epithelium. Activating mutations in KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16 INK4A, p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating KrasG12D mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate KrasG12D and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;KrasG12D or Pdx1-Cre;Smad4lox/lox mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16Ink4A expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated Kras G12D allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4lox/lox animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple KrasG12D-dependent disease-associated phenotypes. ©2007 American Association for Cancer Research.