Heavy carbon-ion beam therapy has revealed several potential advantages over X-rays. Heavy-ion therapy has been applied for various solid tumors including esophageal squamous cell carcinoma (SCC). Although the local control rate in carbon ion radiotherapy for esophageal cancer has revealed better rates than conventional radiotherapy, severe mucosal damage was observed in adjacent normal mucosa. A suitable treatment strategy is required to reduce irradiation dose by introducing combined local therapy. Recently, we initiated clinical p53 gene therapy for esophageal SCC. We herein evaluate the cytotoxic effects of heavy carbon-ion beams combined with p53 gene transfer on human esophageal SCC. We assessed the induction of apoptosis and growth suppression with the use of recombinant adenoviral vector Ad.p53 or heavy carbon-ion beam irradiation or both. Growth suppression was significantly potentiated by combined treatment with heavy carbon-ion beams and Ad.p53 as compared to that treated with either of them alone. Western blot analysis confirmed the expression of both exogenous p53 and p21 proteins after irradiation of Ad.p53 infected cells. Enhanced apoptotic cell death was observed with a terminal deoxynucleotidyl transferase-mediated nick end-labeling assay. These data suggest that heavy carbon-ion beam irradiation combined with Ad.p53 may be a potentially effective therapeutic strategy for locally advanced esophageal SCC.