Tryptophanyl-tRNA Synthetase (WRS) is one of the aminoacyl tRNA synthetase that possess non-canonical functions. Full-length WRS is released during bacterial infection as an important factor priming toll-like receptor 4 (TLR4) - myeloid differentiation factor 2 (MD2) complex to elicit innate immune response. However, role of full-length WRS in virus infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early time of virus infection and functions as an antiviral cytokine. Treatment of recombinant WRS promotes production of inflammatory cytokines and type 1 interferons, and curtails virus replication in THP-1 and Raw264.7 cells. However, we could not observe the similar effects in epithelial cells, TLR4-/- BMDMs, and MD2-/- BMDMs. In vivo, intravenous and intranasal administration of recombinant WRS provoke innate immunity and block vesicular stomatitis virus replication. These findings suggest that secreted full-length WRS has a non-canonical function in activating and mediating innate immune responses to virus infection as well as to bacterial infection. [The Ministry for Food, Agriculture, Forestry and Fisheries (Grant No. 315044031, 316043-3)]