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To study the effect of NS1 eIF4GI binding domain on virulence and pathogenicity of H5N1 influenza A virus,5 recombinant H5N1 viruses encoding eIF4GI binding domain-truncated NS1 proteins and parental NS1 (NS1-wt) were generated by an 8-plasmid-based reverse genetics system.The results indicated that the recombinants with the addition of 5-amino acid and the deletion position of 85-89 in NS1-wt were attenuated in replication in vitro and in vivo,compared with the recombinant wild-type virus rNS1-wt,whereas the deletion position 85-94 or the entire eIF4GI binding domain in NS1-wt displayed a significantly attenuated phenotype in chicken and mice.