A multibasic cleavage site (MBCS) in the hemagglutinin (HA) protein of influenza A virus is a key determinant of pathogenicity in chickens, and distinguishes highly pathogenic avian influenza (HPAI) viruses from low pathogenic avian influenza (LPAI) viruses. A MBCS has only been detected in viruses of the H5 and H7 subtypes. Here we investigated the phenotype of a human H3N2 virus with a MBCS in HA. Insertion of a MBCS in the H3N2 virus resulted in cleavage of HA and efficient replication in MDCK cells in the absence of exogenous trypsin in vitro, similar to HPAI H5N1 virus. However, studies in ferrets demonstrated that insertion of the MBCS in HA did not result in increased virus shedding, cellular host range, systemic replication, or pathogenicity as compared to wildtype virus. This study indicates that acquisition of a MBCS alone is insufficient to increase pathogenicity of a prototypical seasonal human H3N2 virus.