Influenza A virus NS1 protein has developed two main IFN-antagonizing mechanisms by inhibiting retinoic-acid-inducible gene I (RIG-I) signal transduction, or by suppressing cellular pre-mRNA processing through binding to cleavage and polyadenylation specific factor 30 (CPSF30). However, the precise effects of NS1 on suppressing type I IFN induction have not been well characterized. Here we report that compared with PR/8/34 NS1, which is localized partially in the cytoplasm and has strong IFN-antagonizing ability via specifically inhibiting IFN-β promoter activity, H5N1 NS1 has strikingly different characteristics. It mainly accumulates in the nucleus of transfected cells and exerts rather weak IFN-counteracting ability through suppression of the overall gene expression. The M101I mutation of H5N1 NS1, namely H5-M101I, fully reversed its functions. H5-M101I gained the ability to specifically inhibit IFN-β promoter activity, translocate to the cytoplasm, and release CPSF30. The previously reported NES (nuclear export signal) (residues 138-147) was unable to lead H5N1 NS1 to translocate. This suggests that other residues may serve as a potent NES. Findings indicated that together with leucine-100, methionine-101 enhanced the regional NES. In addition, methionine-101 was the key residue for the NS1-CPSF30 interaction. This study reveals the importance of methionine-101 in the influenza A virus life cycle and may provide valuable information for antiviral strategies.