Pancreatic ductal adenocarcinoma (PDAC) is thought to originate from ductal structures, exhibiting strong desmoplastic reaction with stromal pancreatic myofibroblasts (PMF), which are supposed to drive PDAC tumorigenesis. Previously, we observed high expression of the adhesion molecule L1CAM (CD171) in PDAC cells accounting for chemoresistance. Thus, this study aimed to investigate whether PMFs are involved in the induction of tumoral L1CAM and whether this contributes to malignant transformation of pancreatic ductal cells and PDAC tumorigenesis. Immunohistochemistry of tissues from chronic pancreatitis specimens revealed considerable L1CAM expression in ductal structures surrounded by dense fibrotic tissue, whereas no L1CAM staining was seen in normal pancreatic tissues. Using the human pancreatic duct cell line H6c7, we show that coculture with PMFs led to a transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of L1CAM expression. Similarly, L1CAM expression increased in monocultured H6c7 cells after administration of exogenous TGF-beta1. Both TGF-beta1- and PMF-induced L1CAM expression were independent of Smad proteins but required c-Jun NH(2)-terminal kinase activation leading to the induction of the transcription factor Slug. Moreover, Slug interacted with the L1CAM promoter, and its knockdown abrogated the TGF-beta1- and PMF-induced L1CAM expression. As a result of L1CAM expression, H6c7 cells acquired a chemoresistant and migratory phenotype. This mechanism of TGF-beta1-induced L1CAM expression and the resulting phenotype could be verified in the TGF-beta1-responsive PDAC cell lines Colo357 and Panc1. Our data provide new insights into the mechanisms of tumoral L1CAM induction and how PMFs contribute to malignant transformation of pancreatic duct cells early in PDAC tumorigenesis.