Since 2000, more and more NS fragment of H5N1 subtype influenza A virus had a unique deletion of nuleotides from 263 to 277. In order to investigate the biological significance of the mutation, four recombinants, RWSN-848, RWSN-m848, RWSN-248 and RWSN-m248, were generated via eight-plasmid reverse genetic system. These reassortants had the same inner gene and outer gene derived from A/WSN/33(H1N1) and A/D/SD/04(H5N1), respectively. But their NS genes were different. RWSN-m248 and RWSN-848 were isogenic with RWSN-248 and RWSN-m848 respectively, except for the NS gene, the formers encoded a mutant NS with a deletion from 263 - 277 nucleotides. All of the four recombinants could grow well in embryonated chicken eggs and had the similar viral titer, EID50 and MDT. Infections were done with the same Multiplicity of infection (MOI, 0.001), and the viruses in supernatants from infected cells were titrated at 12, 24, 36, and 48 h post-infection by plaque assay in fresh MDCK. The results show that four viruses grown well and had the same viral title in Vero cell, a non-IFN-responsive cell system. But in MDCK and COS-1, the cell lines can produce IFN, RWSN-248 and RWSN-m848 had a higher viral titer (two fold) than RWSN-m248 and RWSN-848. It revealed that the deletion of nucleotides of NS from 263 - 277 of H5N1 influences decreases viral growth ability in IFN producing cell line. IVPI were done for the virulence test of reassorted H5N1 viruses. Ten six-week-old SPF White Leghorn chickens were inoculated intravenously with 0.2mL of a 1:10 dilution of allantoic fluid containing each of the four recombinant viruses. The chickens were daily monitored for disease signs and death for 10 days post-inoculation. Finally, IVPI was counted for each virus. RWSN-m248 caused 4 chickens died in 10 days and its index was 1.63. RWSN-248 only caused one chicken died and its index was 0.45. RWSN848 caused 3 chickens died in 10 days and its index was 0.81. RWSN-m848 caused one of the ten chickens died and its' index was only 0.175. The results revealed that the deletion of nucleotides of NS gene from 263 to 277 sites increases H5N1 pathogenesis in chicken.