DEAD (Asp-Glu-Ala-Asp) box RNA helicase 3 (DDX3X) is demonstrated to have crucial functions in the antiviral immune response. To our knowledge, little information focuses on the function of duck DDX3X. In this study, duck DDX3X (duDDX3X) was cloned and its role in the type I interferon (IFN) signaling pathway was investigated using duck embryo fibroblast (DEF) cells. Full-length duDDX3X cDNA encodes 652 amino acid residues and contains a DEADc domain and a HELICc domain. According to tissue distribution analysis, duDDX3X mRNA was widely expressed in different tissues, especially the spleen and the liver. Overexpression of duDDX3X in DEF cells induced IFN-beta by activating transcription factors IRF1 and NF-kappa B. Knockdown of duDDX3X in DEF cells with siRNA significantly reduced IFN-beta expression induced by poly(I:C), a double-stranded RNA (dsRNA) analog. Our results demonstrated that duck DDX3X was involved in the dsRNA-mediated type I IFN signaling pathway in DEF cells. (C) 2020 Elsevier Inc. All rights reserved.