Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to NeuAcα3-sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved residues of HA surface-located amino acids in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell type dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharides binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to NeuGc. Rescued mutant Y161A viruses demonstrated a 5-10 fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.