Cancer immunotherapeutic agents (vaccines) in the form of antigen-loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC-based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC-based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA-silenced IDO expression, were introduced into 4T1 breast tumor-bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen-loaded DCs without IDO silencing and (iii) reduced CD4+ and CD8+ T cell apoptosis. Furthermore, immunization with IDO-silenced DCs enhanced tumor antigen-specific T cell proliferation and CTL activity, and decreased numbers of CD4+CD25+Foxp3+ Treg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC-based cancer immunotherapeutic.