The incidence of pancreatic ductal adenocarcinoma (PDAC) nearly equals its mortality rate, partly because most PDACs are intrinsically chemoresistant and thus largely untreatable. It was found recently that chemoresistant PDAC cells overexpress the Notch-2 receptor and have undergone epithelial–mesenchymal transition (EMT). In this study, we show that these two phenotypes are interrelated by expression of Midkine (MK), a heparin-binding growth factor that is widely overexpressed in chemoresistant PDAC. Gemcitabine, the front-line chemotherapy used in PDAC treatment, induced MK expression in a dose-dependent manner, and its RNAi-mediated depletion was associated with sensitization to gemcitabine treatment. We identified an interaction between the Notch-2 receptor and MK in PDAC cells. MK–Notch-2 interaction activated Notch signaling, induced EMT, upregulated NF-κB, and increased chemoresistance. Taken together, our findings define an important pathway of chemoresistance in PDAC and suggest novel strategies for its clinical attack. Cancer Res; 71(14); 5009–19. ©2011 AACR.