ABSTRACT The role of Cardif-dependent signaling in controlling dengue virus (DENV) infection and regulating type I interferon (IFN) production in vivo was examined in Cardif-deficient mice. DENV RNA levels were significantly elevated in both the serum and lymphoid tissues of Cardif −/− mice at early times compared to those in wild-type animals. Type I IFN production was delayed in these locales of Cardif −/− mice until 18 h postinfection, indicating that Cardif regulates the initial type I IFN response in lymphoid tissues. In contrast, DENV viral loads in nonlymphoid tissues were similar between Cardif −/− and wild-type mice. These results reveal that RNA helicase-mediated sensing acts as a first line of innate defense against DENV infection in vivo and functions in a tissue-dependent manner.