Clade 2.3.2.1 of the H5N1 avian influenza virus (AIV) evolved into several subclades. However, the effect of glycosylation on the biological characteristics of hemagglutinin (HA) and/or neuraminidase (NA) from H5N1 AIVs remains unclear. Here, we determined that the global prevalence of clade 2.3.2.1 H5N1 AIVs with deglycosylated residue 158 on HA (HA158-) and glycosylated residue 88 on NA (NA88+) were predominant via multiple sequence analysis. The deglycosylation of residue on NA 88 (NA88-) was observed in clade 2.3.2.1a (new) and clade 2.3.2.1e H5N1 AIVs. Interestingly, NA88- was coupled with the acquisition of 158 glycosylation sites on HA (HA158+) in clade 2.3.2.1e H5N1 AIVs from China, and clade 2.3.2.1a (new) H5N1 AIVs exhibiting the HA158-NA88- pattern were predominant in Bangladesh. Meanwhile, the temporal distribution of strain HA158+ NA88- was highly consistent with the implementation of Re-6 vaccine in China. The recombinant H5N1 AIVs constructed using a reverse genetic system showed that the acquisition of the HA158 glycosylation site facilitated viral evasion form Re-6 antisera, and the virus lacking glycosylation sites at HA158 and NA88 resulted in reduced NA activity, replication in mammalian cells, and pathogenicity in both chickens and mice compared to that of the viruses with alternative glycosylation patterns. Therefore, the acquisition of HA158+ in clade 2.3.2.1e H5N1 AIVs enables evasion of Re-6 vaccination pressure, and the virulence of clade 2.3.2.1 H5N1 AIVs is modulated by the absence of glycosylation sites at HA158 and NA88. Our finding highlighting the importance of epidemiological surveillance and timely updating vaccines of H5 AIVs.