The WW-domain-containing oxidoreductase (WWOX) gene spans the common chromosomal fragile site FRA16D (16q23.2) and is believed to be a tumor suppressor in various human malignancies. We have previously shown frequent down-modulation of Wwox expression in pancreatic carcinoma (PC); however, biological function of Wwox in pancreatic duct carcinogenesis remains unknown. In PANC-1 (Wwox-negative) PC-derived cells, restoration of recombinant WWOX gene expression with adenoviral gene delivery (Ad-WWOX) effectively increased the number of cells with subG(1) DNA contents in a multiplicity of infection-dependent manners: Ad-WWOX infection up-regulated caspase-3 activity and reduced procaspase-3 and procaspase-8 levels. We also confirmed that restoration of WWOX gene suppressed cell growth in vitro and tumorigenicity in vivo. In addition, transduction of wild-type WWOX-expressing vector inhibited PANC-1 colony formation; however, substitution of Y33 of Wwox with arginine did not lead to inhibition of colony formation, suggesting the biological significance of the WW1 domain of Wwox for its tumor-suppressing activity. In PC tissue samples, abundant cytoplasmic Wwox expression was detected in the normal pancreatic duct epithelium, whereas Wwox expression was frequently reduced not only in a large fraction of PC but also in precancerous lesions in accord with the pancreatic intraepithelial neoplasia (PanIN) grade, which was closely correlated with patients' poorer outcome. Interestingly, the existence of Wwox expression was associated with elevated mothers against decapentaplegic homolog 4 (Smad4) protein levels in vitro and in vivo. These findings suggest that down-modulation of Wwox expression is an early event and may be associated with the down-regulation of Smad4 protein levels during pancreatic duct carcinogenesis.