The nonstructural protein NS1 of influenza A virus blocks the development of host antiviral responses by inhibiting polyadenylation of cellular pre-mRNA. NS1 also promotes the synthesis of viral proteins by stimulating mRNA translation. Here, we show that recombinant NS1 proteins of human pandemic H1N1/2009, avian highly pathogenic H5N1, and low pathogenic H5N2 influenza strains differentially affected these two cellular processes: NS1 of the two avian strains, in contrast to NS1 of H1N1/2009, stimulated translation of reporter mRNA in cell-free translation system; NS1 of H5N1 was an effective inhibitor of cellular pre-mRNA polyadenylation in A549 cells, unlike NS1 of H5N2 and H1N1/2009. We identified key amino acids in NS1 that contribute to its activity in these two basic cellular processes. Thus, we identified strain-specific differences between influenza virus NS1 proteins in pre-mRNA polyadenylation and mRNA translation.