Type I IFNs shape innate and adaptive immune responses by upregulating the expression of antiviral and immunomodulatory genes. Pathogenic viruses have evolved effective mechanisms to inhibit the type I IFN response, and IAVs are no exception :IAVs express NS1, a multifunctional protein that downregulates type I IFN production. Using site-directed mutagenesis and reverse genetics we characterize the contributions of both strictly conserved and strain-dependent residues in the NS1 putative SH2-binding domain (Y84) and the CPSF4-binding domain (F103 and M106), respectively, to NS1-mediated inhibition of type I IFN signaling and ISG expression. Specifically, we generated rIAVs expressing NS1s encoding Y84F or F103L and/or M106I mutations. We provide evidence that a Y84F mutation abrogates NS1-mediated inhibition of IFN-β-inducible STAT1/2 phosphorylation and IFNAR1 expression. As a result, in human A549 lung epithelial cells and in C57BL/6 mice, rIAVs expressing NS1-Y84F replicate to lower titers and are more susceptible to the antiviral effects of IFN- treatment. rIAVs expressing NS1-F103L/M106I/F103L+M106I are also attenuated in A549 cells. Notably, in A549 cells infected with rIAV expressing NS1-M106I/F103L+M106I and treated with IFN- we identified increased polysome-associated ISG expression – indicative of increased translation initiation – compared with cells infected with rIAV expressing wt NS1. Viewed altogether, our data highlight the ability of IAV NS1 to antagonize multiple aspects of an effective host type I IFN response, and the importance of both conserved and strain-dependent residues in NS1 for IAV replication.