The transcription factor Forkhead box O3 (Foxo3) has a critical role in suppressing the expansion of antigen-specific effector T-cell populations; hence, Foxo3 is a potential target for enhancing the antitumor immunity of cancer vaccines. In this report, we evaluated the potential of RNA interference (RNAi)-mediated silencing of Foxo3 in antigen-presenting cells as an adjuvant for HER2/neu DNA cancer vaccines. Bicistronic plasmids expressing the N-terminal extracellular domain of human HER-2/neu and the Foxo3 short hairpin RNA (hN'-neu-Foxo3 shRNA) or the scrambled control (hN'-neu-scramble shRNA) were subcutaneously injected into mice by gene gun administration to elicit antitumor immunity against p185neu-overexpressing MBT-2 bladder tumor cells. We found that mice treated with hN'-neu-Foxo3 shRNA showed greater reductions in tumor growth and longer survival times than mice treated with hN'-neu-scramble shRNA, indicating that the silencing of Foxo3 enhanced the antitumor efficacy of the HER-2/neu cancer vaccine. Cytotoxicity analyses further revealed that the Foxo3 shRNA-enhanced antitumor effect was associated with significant increases in the number of functional CD8(+) T cells and in the levels of cytotoxic T lymphocytes activity. Interleukin-6 was induced by hN'-neu-Foxo3 shRNA treatment but did not have a critical role in the antitumor effect of the hN'-neu-Foxo3 shRNA vaccine. Moreover, in vivo lymphocyte depletion analyses confirmed that the antitumor efficacy of the hN'-neu-Foxo3 shRNA vaccine depended on functional CD8(+) T cells. Finally, Foxo3 suppression was shown to markedly improve the effect of the HER-2/neu DNA vaccine in limiting the growth and lung metastases of MBT-2 cells. Overall, these results support RNAi-mediated silencing of Foxo3 as an effective strategy to enhance the therapeutic antitumor effect of HER-2/neu DNA vaccines against p185neu-positive tumors.