Attempts have been made to visualize tumor cells intraoperatively with fluorescence guidance. However, the clear demarcation and complete tumor resection have always been a challenging task. To address this, we have developed a novel fluorescence bioimaging system with vesicular stomatitis virus (VSV) incorporating Katushka, near-infrared fluorescent protein. VSV is tumor-specific owing to the deficiency of antiviral interferon signaling pathways in tumor cells. We aimed to evaluate the tumor specificity of the recombinant VSV-Katushka (rVSV-K) in osteosarcoma cells and to assess the feasibility of complete tumor resection by the rVSV-K fluorescence guidance. In in vitro experiments, mouse and human osteosarcoma cell lines and normal human mesenchymal stem cells were infected with rVSV-K and observed by fluorescence microscopy. Near-infrared fluorescence was observed only in osteosarcoma cells, even at a low-concentration of virus infections. In in vivo experiments, mouse osteosarcoma (LM8) cells were transplanted subcutaneously into the back of immune-competent mice to produce an osteosarcoma, which was then injected with rVSV-K. The areas emitting fluorescence were resected using a bioimaging system. The distance between the surgical and tumor margins of the fluorescence-guided resection with rVSV-K group was significantly larger than that of the non-guided resection groups. The local recurrence rate was significantly lower in the fluorescence-guided resection with rVSV-K group than in the non-guided resection groups. The distant metastasis rate and average survival rate were not significantly different between all groups. These results suggest that the rVSV-K is specific to osteosarcoma cells and enables complete tumor resection of osteosarcomas in mice. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.