Abstract Purpose: Tumor-initiating cells with stem-like properties, also termed cancer stem cells (CSC), have been shown to sustain tumor growth as well as metastasis and are highly resistant to chemotherapy. Because pancreatic CSCs have been isolated on the basis of EpCAM expression, we investigated whether a targeted immunotherapy to EpCAM using the bispecific T-cell–engaging antibody MT110 is capable of eradicating CSCs. Experimental Design: We studied in vitro and in vivo the effects of MT110 on CSCs using both established cell lines as well as primary cells of human pancreatic cancer. Results: Although established cell lines were more responsive to MT110-engaged T cells, also primary cells showed a time- and dose-dependent response to treatment with the bispecific antibody. In addition, the population of highly tumorigenic CSCs was efficiently targeted by the EpCAM/CD3-bispecific antibody MT110 in vitro and in vivo using a mouse model of established primary pancreatic cancer. Pancreatic cancer cells derived from metastases were slightly more resistant to MT110 treatment on the basis of in vivo tumorigenicity studies. This appeared to be related to a higher frequency of an EpCAM-negative subpopulation of CSCs. Conclusions: Cytotoxic T cells can be effectively redirected against primary human pancreatic cancer cells by T-cell–engaging BiTE antibody MT110 including a subpopulation of highly tumorigenic CSCs. Clin Cancer Res; 18(2); 465–74. ©2011 AACR.