Toll-like receptor 3 (TLR3) is a significant member of the TLR family which plays a vital role in pathogen-associated molecular patterns recognition and activation of innate immunity. In the past few years, TLR3 has been identified in a variety of fish, but its upstream adaptor is rarely reported. In this study, an adaptor protein, called WDFY1 (WD repeat and FYVE domain-containing protein 1), was cloned and identified in grass carp (Ctenopharyngodon idella) (CiWDFY1). Phylogenetic analysis revealed that fish WDFY1s are highly conserved and share a high level of sequence similarity to their mammalian counterparts. Particularly, CiWDFY1 is clustered in the same clade with that of crucian carp (Carassius auratus) and prussian carp (Carassius gibelio). CiWDFY1 is ubiquitously expressed in all detected tissues (eyes, intestines, gills, skin, liver, spleen, and kidney), but its expression is upregulated upon stimulation with poly(I:C). In CIK cells, over-expression of CiWDFY1 increases, while CiWDFY1 knockdown decreases the expression of IFN1. Subcellular localization assay revealed that CiWDFY1 is evenly distributed throughout the cytoplasm, and yet it is congregated near the cytoplasmic membrane after poly(I:C) treatment. Furthermore, co-immunoprecipitation and co-localization assays demonstrated that CiWDFY1 interacts with TLR3, and then TLR3 recruits TRIF and CiWDFY1 to form an activated complex, resulting in the phosphorylation and nuclear translocation of IRF3. However, CiWDFY1 knockdown hinders the physical interaction between TLR3 and TRIF, as well as the expression of IFN1. Taken together, CiWDFY1 serves as a scaffold protein to induce the expression of IFN1 in a TLR3-TRIF-IRF3-dependent pathway.