Zika virus (ZIKV) nonstructural protein 5 (NS5) is a multifunctional protein possessing methyltransferase and RNA-dependent RNA polymerase activities. In the present study, we have carried out an extensive mutagenesis analysis to determine the importance of nuclear localization sequences (NLS) of NS5 in its nuclear accumulation and ZIKV replication. Deletion mutagenesis analysis demonstrated that the bipartite NLS consisting of importin β1 (βNLS) and importin α/β-recognized NLS (α/βNLS) is required for NS5 nuclear accumulation. Deletion of βNLS, α/βNLS, or both as well as RA and RN mutations severely impaired NS5 nuclear import and consequently conferred NS5 degradation. The RA and RN mutations also ablated viral RNA replication and virus production. Treatment of ZIKV-infected cells with importin α/β-NS5 interaction inhibitors ivermectin or 4-HPR resulted in a rapid degradation of NS5 similar to the R393 A/N mutations. Collectively, these findings suggest that NS5 nuclear accumulation protects NS5 from cytoplasmic degradation and therefore is required for viral RNA replication.Copyright © 2019 Elsevier Inc. All rights reserved.