The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza A virus catalyzes viral RNA transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) by adopting different conformations. A switch from transcription to replication occurs at a relatively late stage of infection. We recently reported that the viral NS2 protein, expressed at later stages from a spliced transcript of the NS segment messenger RNA (mRNA), inhibits transcription, promotes replication and plays a key role in the transcription-to-replication switch. In this study, we performed comprehensive functional analyses to elucidate how NS2 promotes viral genome replication. Using a cell-based single-step RNP reconstitution assay, we found that NS2 specifically promotes the first-step vRNA-to-cRNA synthesis. Further investigation revealed that this promotion is tightly associated with the intrinsic properties of the 3´-vRNA promoter. Employing a highly sensitive complementation reporter assay, we demonstrated that NS2 associates more strongly with the vRNA-resident RdRp than the cRNA-resident RdRp. These findings were further validated through in vitro replication analyses. We, therefore, propose that influenza A virus NS2 protein targets vRNA-resident RdRp to drive the transcription-to-replication switch during infection.