More and more H5N1 subtype avian influenza viruses possessing a 15-nucleotide (15-nt) deletion in the viral nonstructural protein (NS) gene from position 263 to 277 have emerged since 2000. In order to investigate the biological significance of this deletion, two pairs of H5N1 reassortants designated as rWSN-SD versus rWSN-mSD and rWSN-YZ versus rWSN-mYZ were generated by reverse genetics technique. These recombinant viruses shared the same inner genes of PB1, PB2, PA, NP, and M from strain A/WSN/33(H1N1) and outer genes of HA and NA from strain A/Duck/Shandong/093/2004 (H5N1) (A/D/SD/04), whereas they bore different NS gene. Recombinant rWSN-SD carried the full sequence NS gene from A/D/SD/04 in the natural state without deletion, whereas rWSN-mSD carried the same NS gene, but with an artificial 15-nt deletion from position 263 to 277. On the other hand, rWSN-YZ contained the NS gene in the natural state with a deletion from A/Duck/Yangzhou/232/2004 (H5N1) (A/D/YZ/04), while rWSN-mYZ bore the same NS gene but with an artificial insertion of 15-nt in site 263-277. All the four reassortants grew well in embryonated chicken eggs with similar mean death time (MDT) and viral titer of EID50 or HA. However, the virulence of these reassortant viruses in chickens and mice was different. Reassortant viruses with deletion in their NS gene (rWSN-mSD and rWSN-YZ) had much higher intraveneous pathogenicity index (IVPI) in chickens and lower MLD50 in mice than their counterparts without the deletion (rWSN-SD and rWSN-mYZ). Furthermore, rWSN-mSD and rWSN-YZ caused significantly more deaths in infected chickens and higher virus titers in tissues of inoculated mice than did rWSN-SD and rWSN-mYZ respectively. Sequence analysis also showed that H5N1 viruses carrying the 15-nt deletion in the NS gene invariably had the D92E shift in their NS1 protein. The results indicated that the 15-nucleotide deletion of NS gene from site 263 to 277 associated with D92E shift in NS1 protein contributes to the virulence increase of H5N1 viruses in chickens and mice.