Humans infected by H5N1 highly pathogenic avian influenza virus (HPAIV) present unusually high serum concentrations of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The HAs of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactosidase of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6 linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses bearing the HA and NA from the H5N1 HPAIV A/Vietnam/1203/2004 in a PR/8/34 backbone, with specificity for SAα2,3, and a mutant virus (Q226L/G228S in the HA) with preferential receptor specificity changed to SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and IFN inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication as shown by infections with UV inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed differently by human cells than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.