RNA binding ability and cellular distribution are important for nonstructural protein 1 (NS1) of influenza A virus to act as a viral regulatory factor to control virus life cycle. In this study, we identified that the N-terminal residues 19-21 of NS1 are a highly conserved motif depending on all the available NS1 full length sequence of H5N1 influenza A virus from NCBI database. Site-directed mutation analysis demonstrated that the R19 residue of NS1 is critical for its RNA binding and nuclear localization. Furthermore, the residue R19 of NS1 was identified to be critical for regulating M1 mRNA splicing and NS1 nuclear export. Biological analysis of the rescued viruses indicated that the R19A mutation of NS1 did not interfere the replication of H5N1 virus during infection and attenuated the virulence of H5N1 virus in mice.