A key step in the activation of canonical Wnt signaling is the interaction between β-catenin and Tcf/Lefs that forms the transcription activation complex and facilitates the expression of target genes. Eukaryotic initiation factor 4A3 (EIF4A3) is an ATP-dependent DEAD box-family RNA helicase and acts as a core subunit of the exon junction complex (EJC) to control a series of RNA post-transcriptional processes. In this study, we uncover that EIF4A3 functions as a Wnt inhibitor by interfering with the formation of β-catenin/Tcf transcription activation complex. As Wnt stimulation increases, accumulated β-catenin displaces EIF4A3 from a transcriptional complex with Tcf/Lef, allowing the active complex to facilitate the expression of target genes. In zebrafish embryos, eif4a3 depletion inhibited the development of the dorsal organizer and pattern formation of the anterior neuroectoderm by increasing Wnt/β-catenin signaling. Conversely, overexpression of eif4a3 decreased Wnt/β-catenin signaling and inhibited the formation of the dorsal organizer before gastrulation. Our results reveal previously unreported roles of EIF4A3 in the inhibition of Wnt signaling and the regulation of embryonic development in zebrafish.