In recent years, clade 2.3.4.4 H5N6 subtype avian influenza virus (AIV) has been predominantly prevalent in poultry flocks in China. During our AIV surveillance in 2018-2019, 6 circulating strains of H5N6 that possess the natural loss of glycosylation site 154 due to N154D mutation in HA2 protein were isolated. In particular, 5 strains simultaneously carried the V202I mutation in NA protein. Based on the paired backbone H5N6 viruses Y6 and RY6, which just diverged in the glycosylation status at site 158 in HA1 protein, 8 reassortants of rY6-154 N/202 V, rY6-154D/202 V, rY6-154 N/202I and rY6-154D/202I plus rRY6-154 N/202 V, rRY6-154D/202 V, rRY6-154 N/202I and rRY6-154D/202I were constructed with different variation patterns at site 154 in HA2 and site 202 in NA. By determining those reassortants in growth performance on cells, plaque-forming ability, heat and low pH stability, and pathogenicity in mammals, the results showed that HA2 N154D and NA V202I could singly or jointly change the viral biological properties both in vitro and in vivo. Additionally, the effect of HA mutation was significantly more robust than that of NA, and the resulting increasing or reducing impact was closely related to the glycosylation at HA1 site 158. The present study provided a reference for further parsing the relevant mechanism of the functional match between HA and NA proteins of the influenza virus.