Osteosarcoma is the most common primary malignant tumor of bone for adolescent or children. The poor prognosis of patients, due to its remote metastasis, has led to the exploration of more effective and less toxic treatments. Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing tumors. Herein, we describe experiments conducted with a fusion gene, immunocasp-6, which was generated by fusing a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A and an active caspase-6 which can directly cleave lamin A leading to nucleus damage inducing programmed cell death. We demonstrated that immunocasp-6 can specifically and efficiently recognize and induce apoptosis in HER2-overexpressing osteosarcoma cells in vitro. The immunocasp-6 was transferred into BALB/c athymic mice bearing human osteosarcoma by i.m. injection of liposome-encapsulated pCMV-immunocap-6. Expression of immunocasp-6 not only strongly inhibited tumor growth and significantly prolonged animal survival, but also greatly prevented tumor metastasis. Our data showed that the immuno-casp-6 can specifically recognize HER2-overexpressing osteosarcoma cells, can also promptly attack their nucleus and induce apoptotic death, suggesting the potential of this strategy for the treatment of human HER2-overexpressing tumors.