NS gene is generally considered to be related to the virulence of highly pathogenic avian influenza virus (AIVs). In recent years, the strains with 5 amino acids added to the 80-84 positions of the NS1 protein has become prevalent in H5N1 subtype AIVs isolated from mammals. However, the pathogenicity and mechanism of this pattern in mammals remain unclear. In this study, H5N1 subtype AIVs without 80-84 amino acids of the NS1 protein (rNSΔ5aa ) and a mutant virus (rNS5aa-R ) with no deletion of 80-84 amino acids of the NS1 protein were used to determine the pathogenicity in mice. Our results showed that rNS5aa-R possessed an enhanced pathogenicity compared with rNSΔ5aa in vivo and in vitro, which was accompanied by high expression of IL-6, MX1, and CXCL10 in murine lungs. Furthermore, we found that rNS5aa-R increased the infection ability to dendritic cells (DCs). Besides, rNS5aa-R enhanced the expression of phenotypic markers (CD80, CD86, CD40, and MHCII), activation marker CD69, inflammatory cytokines (IL-6, TNF-α, and IL-10) and antagonized interferon (IFN-α) of DCs, in comparison to rNSΔ5aa . Moreover, rNS5aa-R induced DCs to quickly migrate into nearby cervical lymph nodes by highly upregulating CCR7, and CD86 showed a high expression on the migrated DCs. We also found that rNS5aa-R -infected DCs significantly promoted the allogeneic CD4+ T cell proliferation. These findings suggested that rNS5aa-R strongly induced the innate immune response compared with the rNSΔ5aa , which is conducive to activate a wide immune response, resulting a strong cytokine storm and causing an enhanced pathogenicity of H5N1 subtype AIVs in mammals.