Tryptophanyl-tRNA synthetase (WRS) is one of the aminoacyl-tRNA synthetases (ARSs) that possesses non-canonical functions. Full-length WRS is released during bacterial infection and primes the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to elicit innate immune responses. However, the role of WRS in viral infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early phase of viral infection and functions as an antiviral cytokine. Treatment of cells with recombinant WRS protein promotes the production of inflammatory cytokines and type 1 interferons (IFNs) and curtails virus replication in THP-1 and Raw264.7 cells, but not in TLR4-/- or MD2-/- bone marrow-derived macrophages (BMDMs). Intravenous and intranasal administration of recombinant WRS protein induces an innate immune response and blocks viral replication in vivo. These findings suggest that secreted full-length WRS has a non-canonical role in inducing innate immune responses to viral infection as well as to bacterial infection.IMPORTANCE ARSs are essential enzymes in translation that link specific amino acids to their cognate tRNAs. In higher eukaryotes, some ARSs possess additional, non-canonical functions in the regulation of cell metabolism. Here, we report a novel non-canonical function of WRS in antiviral defense. WRS is rapidly secreted in response to viral infection and primes the innate immune response by inducing the secretion of pro-inflammatory cytokines and type 1 IFNs, resulting in the inhibition of virus replication both in vitro and in vivo. Thus, we consider WRS to be a member of the antiviral innate immune response. The results of this study enhance our understanding of host defense systems and provide additional information on the non-canonical functions of ARSs.