Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-positive tumors. Previously, we constructed an immuno-carboxy terminal fragment of Bid (immuno-tBid) and reported its specific and effective destruction of HER2-positive tumor cells. In this study, in order to further reduce the immunogenicity of the previous immuno-proapoptotic protein, we constructed a novel immuno-tBid by replacing domain II of Pseudomonas exotoxin A with a short furin cleavage sequence from the diphtheria toxin. In order to explore the possible application of this novel immuno-tBid in the treatment of osteosarcoma, we first examined the expression of the HER2 protein in a subclone of a human osteosarcoma cell line with relatively high metastatic potential (SOSP-9607-E10), as well as in clinical specimens of osteosarcoma. Quantitative real-time PCR and Western blot analysis revealed that the expression of HER2 was up-regulated in the SOSP-9607-E10 cells, while immunohistochemical analysis revealed that HER2 was overexpressed in 37% of the tissue specimens examined. Both HER2-positive SOSP-9607-E10 and SKBR-3 cells, as well as HER2-negative HeLa cells were transiently transfected with the novel immuno-tBid in order to study its specific pro-apoptotic effect. We demonstrate here that this novel immuno-tBid induces the specific destruction of HER2-overexpressing SOSP-9607-E10 cells through the release of cytochrome C. These results suggest that the novel immuno-tBid with a minimized exogenous fragment could represent a competitive approach for the treatment of HER2-positive osteosarcoma.