An arbovirus of global medical concern, West Nile Virus (WNV) is a positive sense RNA virus from the family Flaviviridae. WNV replication has been extensively studied and been found to occur within the cytoplasm, in conjunction with the modulation of the intracellular membranous architecture. Here we show for the first time that Non-structural protein 5 (NS5) undergoes nucleocytoplasmic shuttling in order to facilitate viral replication, in addition to its known functions as the viral RNA dependent RNA polymerase & methyltransferase. When treated with nuclear export inhibitor Leptomycin B (LMB), an accumulation of NS5 within the nucleus is evident in infected and NS5 transfected cells – demonstrating the ability of NS5 to translocate to the nucleus during infection Through bioinformatics analysis, and using site-directed mutagenesis we have identified a functional nuclear localisation sequence (NLS) responsible for WNV NS5 nuclear translocation, with mutations of this NLS resulting in exclusive cytoplasmic accumulation of NS5, even in the presence of nuclear export inhibitors. When nuclear transport of NS5 is inhibited in an NLS-defective infectious clone, we observed an abolition in replication – suggesting that nuclear import of NS5 is essential for the establishment of WNV replication. Thus, our aims have been to disentangle the function of WNV NS5 nuclear localisation. Novel antiviral strategies could potentially be based on this mechanism to aid the development of therapeutic treatments against WNV as well as pan-flavivirus therapeutics.