Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR) are members of the DEAD box helicases, and recognize viral RNA in the cytoplasm, leading to IFN-beta induction through the adaptor IFN-beta promoter stimulator-1 (IPS-1) (also known as Cardif, mitochondrial antiviral signaling protein or virus-induced signaling adaptor). Since uninfected cells usually harbor a trace of RIG-I, other RNA-binding proteins may participate in assembling viral RNA into the IPS-1 pathway during the initial response to infection. We searched for proteins coupling with human IPS-1 by yeast two-hybrid and identified another DEAD (Asp-Glu-Ala-Asp) box helicase, DDX3 (DEAD/H BOX 3). DDX3 can bind viral RNA to join it in the IPS-1 complex. Unlike RIG-I, DDX3 was constitutively expressed in cells, and some fraction of DDX3 is colocalized with IPS-1 around mitochondria. The 622-662 a.a DDX3 C-terminal region (DDX3-C) directly bound to the IPS-1 CARD-like domain, and the whole DDX3 protein also associated with RLR. By reporter assay, DDX3 helped IPS-1 up-regulate IFN-beta promoter activation and knockdown of DDX3 by siRNA resulted in reduced IFN-beta induction. This activity was conserved on the DDX3-C fragment. DDX3 only marginally enhanced IFN-beta promoter activation induced by transfected TANK-binding kinase 1 (TBK1) or I-kappa-B kinase-epsilon (IKKepsilon). Forced expression of DDX3 augmented virus-mediated IFN-beta induction and host cell protection against virus infection. Hence, DDX3 is an antiviral IPS-1 enhancer.