Respiratory syncytial virus (RSV) is a major global pathogen, causing morbidity and mortality within paediatric populations through pneumonia and bronchiolitis. The virion surface glycoprotein F is an essential player in virus entry into cells, mediating virion-cell membrane fusion and then subsequent cell-cell fusion resulting in virus spread through multinucleate syncytia. During the fusion process, F transitions from a metastable pre-fusion state into a highly stable post-fusion form, stabilised by two heptad repeats (HR) – HRA and HRB. In this study, a third uncharacterised heptad repeat, HRC, was investigated. An alanine scan was performed throughout the HRC helix from Lys-75 to Met-97 in an expression plasmid encoding codon-optimized recombinant RSV F. Epithelial cells were transfected and F protein-mediated cell-cell fusion was assessed by two distinct fusion assays. Protein expression, processing, cell surface representation and protein confirmation were assessed through western blot and flow cytometry. Seven of the mutants completely abrogated F-mediated fusion, eight displayed reduced fusion and six mutants increased cell-cell fusion. When these phenotypes were mapped to the published pre-fusion structure, a striking pattern was observed in that mutants that enhanced fusion aligned on one helical face, while mutants that abrogated fusion lay on another. It is clear that interactions made by residues of HRC play a pivotal role in the fusion process and it is postulated that HRC is crucial in maintaining the delicate stability of the pre-fusion form. The clustering of these residues and proposed mechanism of action provide opportunities that will direct further research and insights that may prove beneficial to anti-viral drug and vaccine design.