Intracellular interferons (IFNs) exert biological functions similar to those of extracellular IFNs, but the signal transduction pathway triggered by the intracellular ligands has not been fully revealed. We investigated the signaling cascade by sequence-specific knockdown of signaling molecules by means of the RNA interference. Truncated IFN-beta gene was constructed so that the N-terminal secretory signal sequence was deleted (SD.IFN-beta). Cells transfected with this construct showed phosphorylation and activation of the STAT1 without any detectable secretion of the cytokine. The MHC class I expression was significantly augmented, while the augmentation was suppressed by short interfering RNA duplexes specific for JAK1, TYK2, and IFN-alpha/beta receptor (IFNAR) 1 and 2c chains. The SD.IFN-beta also induced p53 and phosphorylation of p53 at Ser(15). Specific silencing of p53 abrogated the antiviral effect of SD.IFN-beta, suggesting that the tumor suppressor is critically involved in antiviral defense mediated by intracellular IFN.