Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, and even under optimal therapy these patients face a poor prognosis. Here we report a novel gene therapy-based strategy to battle this disease. We show that the majority of pancreatic tumors overexpress c-erb-B2, which therefore might serve as a target for novel therapies. On the basis of these findings, we developed an adenoviral vector [Ad-e23(scFv)-PE40] encoding a c-erb-B2 (Her-2/neu)-targeted immunotoxin. To improve viral gene delivery we coinfected the therapeutic adenovirus with a replication-competent adenovirus (RCAd) at low doses that enhanced the transduction efficiency of the former virus. In addition, we show that target gene expression can be enhanced by adding etoposide (VP16) at nontherapeutic doses. To investigate the therapeutic efficacy of our approach we established a mouse model for advanced pancreatic cancer disease by intraperitoneal injection of pancreatic cancer cell lines, resulting in multifocal peritoneal xenograft tumors. Administration of Ad-e23(scFv)-PE40 in combination with RCAd and VP16 significantly inhibited tumor growth in mice, with no apparent systemic toxicity. In this study we show that c-erb-B2 might be an effective molecular target in the treatment of pancreatic tumors and that coadministration of a therapeutic c-erb-B2-targeted, non-replication-competent adenovirus with an RCAd and VP16 could be a powerful approach to effectively deliver therapeutic genes to tumors. As demonstrated, this strategy can be employed to effectively treat pancreatic cancer in particular, but may be modified to treat other types of cancer as well.