Author Summary The innate immune system is the first line of defense for organisms that possess an adaptive immune system. It allows a rapid immune response upon viral infections, in addition to propagating an antiviral state in neighboring cells. In an attempt to identify new proteins that are involved in antiviral responses, we completed the first genome-wide RNA interference (RNAi) screen by individually silencing the expression of 15,000 human genes to assess their role in the induction of type I interferon-β (IFNB1) upon Sendai virus (SeV) infection. We identified 237 potential modulator genes for which negative or positive actions of gene products were mapped to the different steps of the antiviral responses. Among these genes, we find two members of the WNT family WNT2B and WNT9B that negatively regulate the IFNB1 response. We show that SeV infection induces the secretion of WNT ligands, stabilization of β-catenin (CTNNB1) and decreases expression of IFNB1 in a feedback mechanism. We further demonstrate that inhibition of glycogen synthase kinase 3 (GSK3) reduces the antiviral innate response in a CTNNB1-dependent manner. The findings suggest that drugs targeting this newly identified canonical-like WNT/CTNNB1 signaling pathway may be therapeutically useful to modulate viral replication or virus-induced inflammation.