Gene transfection to cancer cells using nanobubbles and ultrasound (NBs-US) is a non-invasive gene therapy. Herpes simplex virus thymidine kinase (HSVtk) gene is a representative suicide gene, and has been demonstrated to have potential of cytotoxic sensitivity by ganciclovir (GCV). In this report, we evaluated the effectiveness of HSVtk/GCV cytotoxic gene therapy on cancer cells using NBs-US. For in vitro studies, HSVtk was trasnfected into five cell lines (A549, EMT6, colon26, HT29 and 293T) with NBs-US (0.6MPa). The effects of HSVtk/GCV gene therapy were evaluated using an MTT assay. For in vivo studies, HT29 cells expressing luciferase gene were trasnsplanted into mice. HSVtk was transfected into tumor with NBs-US and GCV was administrated intraperitoneally. Luciferase activity regarded as tumor size was measured by a bioluminescence imaging system. Both in vitro and in vivo studies, cytotoxixity was obtained only in the treated cells with HSVtk/GCV. These results demonstrated the potential to treat cancer cells by using the NBs-US method combined with suicide gene therapy.