ABSTRACTHuman influenza viruses evade host immune responses by accumulating mutations around the receptor-binding region of the hemagglutinin (HA) protein, which is composed of three key elements, the 130-loop, the 190-helix, and the 220-loop. Here, we characterized two human H3N2 influenza viruses with 12- and 16-amino acid deletions around the HA receptor-binding site that were isolated after antigenic selection of mutated H3N2 viruses. Structural modeling suggested that the 12-amino acid deletion eliminated the 190-helix. The 16-amino acid deletion comprises two stretches of 11- and 5-amino acid deletions. As the result of a frameshift, “novel” amino acids (not found in wild-type HA at these positions) are encoded between the deleted regions. Interestingly, structural modeling predicted that the novel sequence forms a structure resembling the 190-helix. However, compared to wild-type HA, the 16-amino acid deletion mutant lacks two antiparallel beta-sheets that connect the 190-helix and the 220-loop in wild-type HA. Nonetheless, both HA deletion mutants replicated in mammalian cells, and the 16-amino acid deletion mutant (with a remodeled 190-helix) also replicated in Syrian hamsters, albeit at low titers. Wild-type virus bound preferentially to α2,6-linked sialic acids, whereas both mutants gained affinity for α2,3-linked sialic acids. Moreover, the 12- and 16-amino acid deletions may affect the antigenic properties of the viruses. Thus, viruses with sizeable deletions around the HA receptor-binding site are viable but may display altered sialic acid preferences, altered antigenic properties, and attenuated replicative ability in cultured cells and virulence in Syrian hamsters.