Unlike pathogens such as bacteria and fungi, the types of glycans on enveloped viruses stem from host biosynthetic pathways and are specific to the cells used during viral replication. Glycosylated surface proteins are incorporated into progeny virions and host cell membranes, and the attached glycans can play multifaceted roles in protein biosynthesis, function and host-virus interactions. These include shielding antigenic epitopes, or acting themselves as epitopes for recognition and neutralisation by the host immune system. The leading vaccine based immunogens for enveloped viruses mimic surface glycoproteins and exhibit native-like antigenic properties1. Efforts to develop the first effective vaccine against respiratory syncytial virus (RSV), an important cause of acute lower respiratory infection in infants and immunocompromised individuals, have focused the fusion (F) surface glycoprotein. However, our glycoproteomic studies of F-based vaccine candidates revealed substantially different glycosylation patterns depending on the cell line used. We compared F produced in adenocarcinomic human alveolar basal epithelial cells and human embryonic kidney cells, and highlighted unique glycan motifs on F after production in the latter cell line. Fragmentation of predominate N-linked glycopeptides produced glycan oxonium ions consistent with two terminal N-acetyl-hexosamine units. These monosaccharide units could potentially represent β1–4-linked N-acetyl-galactosamine and N-acetyl-glucosamine, commonly called LacdiNAc motifs. These motifs have been identified on a relatively small number of mammalian proteins and have been shown to provoke humoral immune responses and exhibit immunosuppressive activities. Previous structural predictions of F identified an N-linked sequon within an antigenic site targeted by potent neutralising antibodies2, 3. Thus, F-based vaccines with N-glycans containing LacdiNAc motifs may not generate protective antibodies after administration, or may prime aberrant immune responses if the epitopes are not present during natural infection. This work highlights the importance of cell-line choice when producing therapeutics to protect against RSV infection.