The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN-α did not show cross-species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN-α effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN-α augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN-α gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN-α adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM-1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell- and natural killer cell-mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM-1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN-α gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity.