Flaviviruses such as West Nile (WNV), Zika (ZIKV) and yellow fever (YFV) viruses pose continuing risks to public health. Currently specific treatments for flavivirus infections are not available, which is partly due to our limited understanding of the mechanisms of virus-host interactions. tRNA-halves (tiRNA) are small non-coding RNAs generated from mature tRNAs via nucleolytic cleavage in the anti-codon loop producing two halves (5p and 3p). The function of tiRNAs is an emerging research field with currently, limited understating of their roles in response to viruses. Herein we demonstrate that flaviviruses including WNV, ZIKV and YFV induce tiRNA production during infection and show their involvement in viral replication and host responses. Using small RNA-Seq we discovered that WNV infection increased accumulation of small 30-32nt RNAs that primarily consist of tiRNAs. The most induced tiRNAs were Glu-CTC and SecA 5p-fragments. We validated induction of these tiRNAs using northern blotting and ascertained that ZIKV and YFV also induced their production. We found that contrary to previous reports that implicated angiogenin in production of tiRNAs, this enzyme was not expressed in our model. However, the orthologous RNase 4 was induced upon viral expression as evident by RNA-Seq and qRT-PCR. Moreover, RNAi knock-down of RNase4 significantly reduced tiRNA production in infected cells and attenuated viral replication, which indicates for the requirement of this RNase for virus-induced tiRNA generation. In addition, we demonstrated that the recombinant RNase4 can generate tiRNAs in vitro. To assess the effect of tiRNAs on viral replication we transfected cells with synthetic tiRNA mimics and antisense RNA oligonucleotides followed by WNV infection and virus titration. This revealed the potential proviral role of tiRNAs in infected cells. In summary, our findings indicate that tiRNA halves are produced in response to flavivirus infection and play an important role in the viral replication cycle.