In clinical N0 (cN0) cases with head and neck squamous cell carcinoma (HNSCC), a treatment selection is still controversial: elective neck dissection or watchful waiting. We focused on sentinel lymph node (SLN)-targeted therapy using the urokinase-type plasminogen activator (uPA)-dependent oncolytic Sendai virus "BioKnife." The objectives of this study were to investigate BioKnife migration into SLNs and elucidate its antitumor effect on lymph node metastases (LNM). We established an orthotopic nude mouse model of HNSCC, with LNM being frequently induced. We inoculated HSC-3-M3, human highly metastatic tongue squamous cell carcinoma cells, in the tongue of the nude mice, and after 2 weeks, we injected BioKnife into the primary tumor. We tracked BioKnife migration into the SLNs by immunostaining, RT-PCR, and an in vivo imaging system. We also examined its antitumor effects and mechanisms through serial section analysis of lymph nodes. GFP reporter expression was clearly visible in the lymph nodes of virus groups, which corresponded to SLNs. Relative GFP mRNA was significantly increased in both the tongues and lymph nodes in the virus groups compared with that in the control group (P < 0.05). Serial section analysis showed that BioKnife infected cancer cells and exhibited significant antitumor effect against LNM compared with the control groups (P < 0.05). We detected apoptosis in LNM infected by BioKnife. BioKnife migrated into SLNs after its injection into the primary tumor and effectively suppressed LNM, suggesting that SLN-targeted therapy using BioKnife has great potential to provide a novel and promising alternative to elective neck dissection in cN0 patients with HNSCC.