We previously reported the selective killing of HER2-positive tumor cells by a class of immunoproapoptotic proteins containing single-chain antibody, translocation domain of Pseudomonas exotoxin A (domain II; PEA II), and constitutively active human apoptotic molecules. In this study, a novel class of antitumor immunoproapoptotic proteins was explored to mediate tumor-specific apoptosis both in vitro and in vivo. Three furin cleavage sequences, including a synthetic polyarginine tract, and two furin cleavable sequences from PEA and diphtheria toxin were respectively used to replace PEA II in the previously constructed immunoproapoptotic protein. When produced and secreted by the genetically modified Jurkat cells, the novel targeted proapoptotic proteins selectively bound to HER2, which is often overexpressed on tumor cell surface. Followed by receptor-mediated endocytosis and furin cleavage in the endosome, the recombinant proteins could translocate into the cytosol, leading to irreversible cell death. Moreover, delivery of these proteins by either i.m. plasmid injection or i.v. injection of plasmid-expressing Jurkat cells led to tumor regression and prolonged animal survival in a nude mouse xenograft tumor model, indicating in vivo antitumor activity of the recombinant proteins. We conclude that the new class of immunoproapoptotic proteins show comparable activity with PEA II-containing counterpart and provide an attractive therapeutic alternative as they contain much less exogenous fragments.