The nucleoprotein (NP) of influenza A virus is transported into the nucleus via the classical importin α/β pathway, and proceeds via nuclear localization signals (NLSs) recognized by importin α molecules. Although NP binds to importin α isoforms Rch1, Qip1 and NPI-1, the role of each individual isoform during the nuclear transport of NP and replication of the influenza virus remains unknown. In this study, we examined the contribution of importin α isoforms for nuclear localization of NP and viral growth using a panel of NP mutants containing serial alanine replacements within an unconventional NLS of NP. Alanine mutation at amino acid 8 (R8A) caused a significant reduction in the nuclear localization and binding to the three importin isoforms. The R8A NP mutant virus did not generate by reverse-genetics approach. This indicates that position 8 is the main site that mediates nuclear localization via interactions with Rch1, Qip1 and NPI-1, and subsequent viral production. This was confirmed by the finding that the conservation of amino acid 8 in human- and avian-origin influenza virus NP was necessary for virus propagation. By contrast, another mutant, S9A NP, which localized in the nucleus, caused a reduction in viral growth and vRNA transcription, suggesting that the unconventional NLS within NP may be associated with nuclear transport, vRNA transcription and viral replication through independent pathways. Interestingly, the N-terminal 110-amino acid region, which contained the unconventional NLS with S9A mutation, mainly bound to Qip1. Furthermore, activities of vRNA transcription and replication of S9A NP mutants were decreased by silencing Qip1 in without changing nuclear localization, indicating that Qip1 involves in multiplication of S9A mutant virus independently of nuclear transport function. Collectively, our results demonstrate the unconventional NLS within NP might have the additional ability to regulate the viral replication that is independent of nuclear localization activity via interactions with Qip1.