Pancreatic cancer is characterized by a dense fibrotic stroma, consisting of cancer-associated fibroblasts (CAFs), immune cells, and extracellular matrix (ECM) that promote tumor growth and treatment resistance. CAFs secrete and organize on their surface ECM proteins, including fibronectin (FN) which coordinates the assembly of collagen fibrils that promote matrix stiffness. Since CAFs are a major contributor to fibrosis and the progression of pancreatic cancer, novel approaches to target their function could have a significant impact on patient outcome. This research demonstrates that the ability of pancreatic cancer cells to initiate tumors in mice is greatly enhanced by co-injection of CAFs. Targeting FN via FN-binding integrins α5β1 and αvβ3 that are highly expressed on CAFs disrupts CAF-induced fibrosis, decreases tumor stiffness, and prevents the initiation of pancreas tumors in mice. These results highlight the therapeutic potential of targeting fibronectin-binding integrins, α5β1 and αvβ3, on CAFs to disrupt the fibrotic stroma in pancreatic cancer.