Updated project metadata. During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitin landscape of the IAV polymerase during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG- (di-glycyl)-remnant-bearing peptides. We identified 59 modified lysines across all three subunits of the viral polymerase of which 17 distinctively affected mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, our results demonstrate that the ubiquitinated residue K578 in the PB1 thumb domain is crucial for the dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R impeded the steps of cRNA stabilization and vRNA transcription, respectively, and affected NP binding as well as polymerase dimerization. Collectively, our results indicate that ubiquitin-mediated disruption of the charge-dependent interaction between PB1-K578 and PB2-E72 is required to coordinate polymerase dimerization and facilitate vRNA replication, which demonstrates that IAV exploit the cellular ubiquitin system to modulate the activity of the viral polymerase for the regulation of viral replication.