RNA interference (RNAi), which causes the degradation of any RNA in a sequence specific manner, is a posttranscriptional gene silencing mechanism. Targeting the invariant chain (Ii) in DCs has been used as an approach to enhance antitumor immunity. It is demonstrated in this article that transfection of H-2(K) DCs with siRNA specific for Ii gene can significantly knock down Ii. When exposed to TNF-alpha, immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production. Ii siRNA-treated H-2(K) DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay. Furthermore, Ii siRNA-transfected H-2(K) DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production, and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro. Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.