The hemagglutinin (HA) protein is a major virulence determinant for the 1918 pandemic virus, however it encodes no known virulence-associated determinants. In comparison to seasonal influenza viruses of lesser virulence, the 1918 H1N1 virus has fewer glycosylation sequons on the HA globular head region. Using site-directed mutagenesis we found that a 1918 HA recombinant virus, of high virulence, could be significantly attenuated in mice by adding two additional glycosylation sites (asparagine [Asn] 71 and Asn 286) on the side of the HA head. The 1918 HA recombinant virus was further attenuated by introducing two additional glycosylation sites on the top of the HA head at Asn 142 and Asn 172. In a reciprocal experimental approach, deletion of HA glycosylation sites (Asn 142 and Asn 177, but not Asn 71 and Asn 104) from a seasonal influenza H1N1 virus, A/Solomon Islands/2006 (SI/06) displayed increased virulence in mice. The addition of glycosylation sites to the 1918 HA and removal of glycosylation sites from SI/06 HA imposed constraints on the theoretical structure surrounding the glycan-receptor binding sites, which in turn led to distinct glycan receptor binding properties. The modification of glycosylation sites for the 1918 and SI/06 virus also caused changes in viral antigenicity based on cross hemagglutinin-inhibition antibody titers with antisera from mice infected with wild type or glycan mutant viruses. These results demonstrate that glycosylation patterns of the 1918 and seasonal H1N1 virus directly contributes to differences in virulence and is partially responsible for their distinct antigenicity.