Glioblastoma multiforme (GM), the most frequent primary malignant brain tumor, is highly invasive due to the expression of proteases, including urokinase-type plasminogen activator (uPA). Here, we show the potential of our new and powerful recombinant Sendai virus (rSeV) showing uPA-specific cell-to-cell fusion activity [rSeV/dMFct14 (uPA2), named "BioKnife"] for GM treatment, an effect that was synergistically enhanced by arming BioKnife with the interferon-β (IFN-β) gene. BioKnife killed human GM cell lines efficiently in a uPA-dependent fashion, and this killing was prevented by PA inhibitor-1. Rat gliosarcoma 9L cells expressing both uPA and its functional receptor uPAR (9L-L/R) exhibited high uPA activity on the cellular surface and were highly susceptible to BioKnife. Although parent 9L cells (9L-P) were resistant to BioKnife and to BioKnife expressing IFN-β (BioKnife-IFNβ), cell-cell fusion of 9L-L/R strongly facilitated the expression of IFN-β, and in turn, IFN-β significantly accelerated the fusion activity of BioKnife. A similar synergy was seen in a rat orthotopic brain GM model with 9L-L/R in vivo; therefore, these results suggest that BioKnife-IFNβ may have significant potential to improve the survival of GM patients in a clinical setting.