The currently circulating influenza B viruses from both antigenic lineages contain an N-linked glycosylation site in the hemagglutinin (HA) protein at positions of 196 or 197. However, egg adaptation caused the loss of the glycosylation site that could impact virus antigenicity and vaccine efficacy. The effect of the 196/197 glycosylation site on influenza B virus growth and antigenicity was systemically evaluated in this study by the molecular approach. Paired recombinant 6:2 reassortant influenza B vaccine strains, with or without the 196/197 glycosylation site, were generated by reverse genetics and the glycosylation site was retained in MDCK cells. In contrast, all the viruses that contained the introduced glycosylation site were unable to grow in eggs and rapidly lost the glycosylation site once adapted to grow in eggs. We showed that glycosylation affected virus binding to the alpha-2,3-linked sialic acid receptor and affected virus antigenicity as tested by postinfected ferret sera. We have further identified that the Arginine residue at amino acid position 141 (141R) can stabilize the 196/197 glycosylation site without affecting virus antigenicity. Thus, the 141R could be introduced into vaccine strains to retain the 196/197 glycosylation site for influenza B vaccines.