Regulatory T cells (Tregs) play an important role in immunological self-tolerance and protect the host from autoimmune disease. However, in cancer immunity, Tregs might block anti-tumor immune responses. Therefore, the depletion of Tregs using a specific agent that suppresses its function or population, such as an anti-CD25 antibody, could promote anti-tumor immune responses. In the present study, a cytotoxicity assay, enzyme-linked immunosorbent spot (ELISPOT) assay and measuring cytokine secretion, were used to study the efficacy of Treg depletion by anti-CD25 antibody added to a dendritic cell/tumor cell (DC/TC) fusion hybrid vaccine in a murine pancreatic cancer model. All the mice treated with the combined therapy of fusion hybrid vaccine and Treg depletion rejected tumor growth in a challenging test, although the rejection rate was 20% both for mice that received the fusion hybrids alone or Treg depletion alone. In addition, combined therapy showed a significantly improved survival in comparison to other treatment or control groups. The NK cell activity for DC/TC fusion + Treg depletion was significantly higher than that for the other treatment groups. Cytotoxic T lymphocyte (CTL) activity for DC/TC could potentially be enhanced by the addition of Treg depletion therapy. The treatments including DC/TC fusion induced IFN-gamma secreting effector cells in ELISPOT assays. Furthermore, a cytometric beads array assay used to measure cytokine secretion showed that DC/TC fusion + Treg depletion stimulated the highest levels of IFN-gamma Th1/Th2 ratios and Th17. These results demonstrate that Treg depletion combined with DC/TC fusion hybrid vaccine enhanced the efficacy of immunotherapy in pancreatic cancer by activating CTLs and NK cells.