Influenza A and B viruses are significant human pathogens responsible for the annual seasonal "flu". Diverged some 2000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and very limited host range. Based on sequence comparison and our prior structural studies, we hypothesized that a key difference in the receptor-binding site of influenza virus hemagglutinin (HA), phenylalaline (Phe) 95 in influenza B virus HA (BHA), versus tyrosine (Tyr) in influenza A virus HA (AHA), is possibly the molecular basis for the different receptor-binding affinity. We further hypothesized that this could be at least partially responsible for the very limited host range of influenza B virus. By using glycan and red blood cell binding assays, we demonstrated that the mutation Phe95[arrow right]Tyr in BHA substantially enhanced receptor-binding affinity. Furthermore, this mutation efficiently competed against the infection of influenza A virus and greatly improved the binding of BHA to three mammalian cell lines. Taken together, residue 95 of BHA appears to be a key determinant for the receptor binding affinity and host range of influenza B virus.