Bovine respiratory syncytial virus (BRSV) is an etiological component of the bovine respiratory tract disease complex. Infection with BRSV following vaccination, or re-infection following natural infection is common since protection is incomplete. The objectives of this study were to create plasmid DNA constructs encoding single or multiple N-glycosylation-site deletion BRSV fusion (F) proteins, and evaluate their expression in cell culture, and potential to induce anti-BRSV F antibody responses in BALB/c mice. Four plasmid DNAs were constructed, each encoding 1-4 N-glycosylation-site deletions: Gly4, Gly2/4, Gly1/2/4 and Gly1/2/3/4. Each of the N-glycosylation-site deletion BRSV F proteins were expressed in COS-7 cells following transfection with plasmid DNA. Inoculation of BALB/c mice with plasmid DNA, resulted in a significant anti-BRSV F IgG response to the wild-type (WT) F and glycosylation-site deletion protein Gly2/4. Gly2/4 elicited a higher antibody titer than the fully glycosylated WT F protein. Significant neutralizing antibody titers were detected following immunization with the Gly2/4 plasmid DNA. These glycosylation-site deletion BRSV F proteins will be useful to characterize the effects of glycosylation on immunogenicity in the natural host, and may lead to a new approach for the generation of BRSV vaccines.