Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Importin-alpha 7 is a component of the nuclear import machinery required for avian-mammalian adaptation and replicative fitness in human cells. Here, we elucidate the mechanisms by which influenza A viruses may escape replicative restriction in the absence of importin-alpha 7. To address this question, we assessed viral evolution in mice lacking the importin-alpha 7 gene. We show that three mutations in particular occur with high frequency in the viral nucleoprotein (NP) protein (G102R, M105K and D375N) in a specific structural area upon in vivo adaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin-alpha 7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal. However, viral escape from importin-alpha 7 by mutations in NP is, at least in part, associated with reduced viral replication highlighting the crucial contribution of importin-alpha 7 to replicative fitness in human cells.