Abstract Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with one of the worst outcomes among all cancers. PDA often recurs after initial treatment to result in patient death despite the use of chemotherapy or radiation therapy. PDA contains a subset of tumor-initiating cells capable of extensive self-renewal known as cancer stem cells (CSC), which may contribute to therapeutic resistance and metastasis. At present, conventional chemotherapy and radiotherapy are largely ineffective in depleting CSC pool, suggesting the need for novel therapies that specifically target the cancer-sustaining stem cells for tumor eradication and to improve the poor prognosis of PDA patients. In this study, we report that death receptor 5 (DR5) is enriched in pancreatic CSCs compared with the bulk of the tumor cells. Treating a collection of freshly generated patient-derived PDA xenografts with gemcitabine, the first-line chemotherapeutic agent for PDA, is initially effective in reducing tumor size, but largely ineffective in diminishing the CSC populations, and eventually culminated in tumor relapse. However, a combination of tigatuzumab, a fully humanized DR5 agonist monoclonal antibody, with gemcitabine proved to be more efficacious by providing a double hit to kill both CSCs and bulk tumor cells. The combination therapy produced remarkable reduction in pancreatic CSCs, tumor remissions, and significant improvements in time to tumor progression in a model that is considered more difficult to treat. These data provide the rationale to explore the DR5-directed therapies in combination with chemotherapy as a therapeutic option to improve the current standard of care for pancreatic cancer patients. Mol Cancer Ther; 9(9); 2582–92. ©2010 AACR.