The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed by PB1, PB2 and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nucleus of infected cells. In this report, we newly identified a host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with PB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by the PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain but not on its typical J-domain, while the J-domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to entre nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner.