Osteosarcoma is the most common primary malignant bone tumor, with high rates of metastasis. Here, we examined the expression of human epidermal growth factor receptor-2 (HER-2) in osteosarcoma cell lines with different metastatic potential, finding that the expression was correlated with metastasis of implanted tumors. We then introduced an expression vector encoding the e23sFv-PEA II-Bid ∆1-60 gene, composed of a HER2-specific single-chain antibody fused with domain II of Pseudomonas exotoxin A (PEA) and the carboxy end of truncated active Bid. We demonstrated that the e23sFv-PEA II-Bid ∆1-60 molecule selectively recognized and killed HER2-overexpressing osteosarcoma cells in vitro. Subsequently, we introduced the e23sFv-PEA II-bid ∆1-60 gene into BALB/c athymic mice bearing HER2-positive osteosarcomas using i.m. injections of liposome-encapsulated vector. Expression of the e23sFv-PEA II-Bid ∆1-60 gene suppressed tumor growth, significantly prolonged animal survival, and inhibited metastasis, thereby suggesting it may represent a competitive approach to treating HER2/neu-positive osteosarcoma.