Influenza A virus (IAV) non-structural protein 1 (NS1) is a protein with multiple functions that are regulated by phosphorylation. Phospho-proteomic screening of H1N1-infected cells identified NS1 to be phosphorylated at serine 205 in intermediate stages of the viral life cycle. Interestingly, S205 is one of six amino acid changes in NS1 of post-pandemic H1N1 viruses currently circulating in humans compared to the original swine-origin 2009 pandemic (H1N1pdm09) virus, suggesting a role in host adaptation. To identify NS1 functions regulated by S205 phosphorylation, we generated both, recombinant PR8 H1N1 NS1 mutants S205G (non-phosphorylatable) and S205N (H1N1pdm09-signature), as well as H1N1pdm09 viruses harboring the reverse mutation NS1 N205S or N205D (phospho-mimetic). Replication of PR8 NS1 mutants was attenuated compared to wild type (WT) in a porcine cell line. However, PR8 NS1 S205N showed a remarkably higher attenuation compared to PR8 NS1 S205G in a human cell line, highlighting a potential host independent advantage of phosphorylatable S205, while an asparagine at this position leads to a potential host-specific attenuation. Interestingly, PR8 NS1 S205G did not show polymerase activity enhancing functions compared to WT, which can be attributed to a diminished interaction with cellular restriction factor DDX21. Analysis of the respective kinase mediating S205 phosphorylation indicated an involvement of CK2. CK2 inhibition significantly reduced replication of WT viruses and decreased NS1-DDX21 interaction as observed for NS1 S205G. In summary, NS1 S205 is required for efficient NS1-DDX21 binding resulting in an enhanced viral polymerase activity, which is likely to be regulated by transient phosphorylation.Importance Influenza A viruses (IAV) still pose a major threat to human health worldwide. As a zoonotic disease, IAV can spontaneously overcome species barriers and even reside in new hosts after efficient adaptation. Investigation of the function of specific adaptational mutations can lead to a deeper understanding of viral replication in specific hosts and can probably help to find new targets for antiviral intervention. In the present study, we analyzed the role of NS1 S205, a phosphorylation site that was re-acquired during circulation of pandemic H1N1pdm09 "Swine flu" in the human host. We found that phosphorylation of human H1N1 NS1 S205 is mediated by cellular kinase CK2 and is needed for efficient interaction with human host restriction factor DDX21 mediating NS1-induced enhancement of viral polymerase activity. Therefore, targeting CK2 activity might be an efficient strategy to limit replication of IAV circulating in the human population.