Since 2003, H5N1-subtype avian influenza viruses (AIVs) with both a deletion of 20 amino acids in the stalk of the neuraminidase (NA) glycoprotein (A-) and a deletion of five amino acids at positions 80 to 84 in the non-structural protein NS1 (S-) have become predominant. To understand the influence of these double deletions in the NA and NS1 proteins on the pathogenicity of H5N1-subtype AIVs, we selected A/mallard/Huadong/S/2005 as a parental strain to generate rescued wild-type A-S- and three variants (A-S+ with a five-amino-acid insertion in the NS1 protein, A+S- with a 20-amino-acid insertion in the NA stalk, and A+S+ with insertions in both NA and NS1 proteins) and evaluated their biological characteristics and virulence. The titers of the AIVs with A- and/or S- replicated in DEF cells were higher than that of A+S+, and the A-S- virus exhibited a replication predominance when co-infected with the other variants in DEF cells. In addition, A-S- induced a more significant increase in the expression of immune-related genes in peripheral blood mononuclear cells of mallard ducks in vitro compared with the other variants. Furthermore, an insertion in the NA and/or NS1 proteins of AIVs resulted in a notable decrease in virulence in ducks, as determined by intravenous pathogenicity index, and the two insertions exerted a synergistic effect on the attenuation of pathogenicity in ducks. In addition, compared with A+S+ and A+S-, the A-S+ and A-S- viruses that were introduced via the intranasal inoculation route exhibited a faster replication ability in the lungs of ducks. These data indicate that both the deletions in the NA stalk and the NS1 protein contribute to the high pathogenicity of H5N1 AIVs in ducks.