Antigen-presenting cells expressing indoleamine 2,3-dioxygenase (IDO) play a critical role in maintaining peripheral tolerance. Strategies to inhibit IDO gene expression and enhance antigen-presenting cell function might improve anti-tumour immunity. Here we have designed highly effective anti-IDO small interfering (si) RNAs that function at low concentrations. When delivered to human primary immune cells such as monocytes and dendritic cells (DCs), they totally inhibited IDO gene expression without impairing DC maturation and function. Depending on the design and chemical modifications, we show that it is possible to design either monofunctional siRNAs devoid of immunostimulation or bifunctional siRNAs with gene silencing and immunostimulatory activities. The latter are able to knockdown IDO expression and induce cytokine production through either endosomal Toll-like receptor 7/8 or cytoplasmic retinoid acid-inducible gene 1 helicase. Inhibition of IDO expression with both classes of siRNAs inhibited DC immunosuppressive function on T-cell proliferation. Immature monocyte-derived DCs that had been transfected with siRNA-bearing 5'-triphosphate activated T cells, indicating that, even in the absence of external stimuli such as tumour necrosis factor-alpha, those DCs were sufficiently mature to initiate T-cell activation. Collectively, our data highlight the potential therapeutic applications of this new generation of siRNAs in immunotherapy.