Cancer-associated fibroblasts (CAFs) contribute to cancer progression that is caused by epithelial-to- mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor-promoting cancer stroma. Here we report that alpha-smooth muscle actin (α-SMA)-positive myofibroblast-like cells originating from MSCs contribute to inducing EMT in Side Population (SP) cells of pancreatic cancer. More importantly, MSCs-derived myofibroblasts function to maintain tumor-initiating stem cell-like cells (TISCs)-like characteristics, including augmenting expression levels of various stemness-associated genes, enhancing sphere- forming activity, promoting tumor formation in a mouse xenograft model, and exhibiting resistance to anticancer drugs. Furthermore, both γ-secretase inhibitor and small interfering RNA (siRNA) directed against Jagged-1 attenuated MSCs-associated E-cadherin suppression and sphere formation in pancreatic cancer SP cells. Thus, our results suggest that MSCs-derived myofibroblasts play important roles in regulating EMT and TISCs-like properties of pancreatic cancer cells through an intermediating Notch signal.