A single intranasal immunization with VSV-GP-Fsyn induces RSV-neutralizing antibodies and reduces RSV viral load by 2 log. BALB/c mice (n = 5) were immunized in weeks 0 and 4 intramuscularly with 107 TCID50 VSV-GP-Fsyn, VSV-GP or 106 formalin inactivated (FI)-RSV, intranasal with 107 VSV-GP-Fsyn or first intramuscular followed by intranasal with 107 VSV-GP-Fsyn. One group received a single intranasal immunization with 107 VSV-GP-Fsyn in week 4. (A) EDTA plasma was collected 4 weeks after prime and boost and analyzed for the titer of RSV neutralizing antibodies; (B) in week 4 after boost, mice were infected with 106 PFU of replication competent RSV. On day 5 after infection, mice were sacrificed, and lungs were analyzed for the titer of RSV by qPCR. Statistical significances were determined in a one-way analysis of variance (Kruskal–Wallis test) followed by Dunn’s Multiple Comparison Test (*, p ≤ 0.05; **, p ≤ 0.01). Geometric mean is indicated; the dotted lines indicate the detection limits of the assays.