Human parainfluenza viruses (hPIV) are pathogens responsible for upper and lower respiratory tract infections. We previously described clinical variant strains of hPIV-2 that display unusual large syncytial cytopathic effects. Their molecular characterization revealed a recurrent conserved specific amino acid substitution: A96T in the F2 subunit of the fusion glycoprotein F. The objective of this study was to investigate the contribution of this A96T substitution to the specific hyperfusogenic properties of the hPIV-2 variant strains. Based on a transient expression strategy, quantification of cell-cell fusion assays, and flow cytometry, we have shown that the A96T mutation strongly alters the fusogenic properties of F hPIV-2, highlighting this key residue in the F2 subunit and its possible role in fusion regulation. This work highlights the benefits of monitoring genetic and phenotypic changes of circulating strains to complete our understanding of Paramyxovirus fusion and related pathogenesis.