The second domain of Pseudomonas exotoxin A (PEAII, residues 253-364) has been shown to facilitate translocation of extracellular and vesicular contents into the cytoplasm, and can transport heterologous molecules into target cells. Because full length PEAII may elicit a host immune response, we tried to identify the minimal PEAII translocation motif and use this fragment in combination with an antibody and constitutively active granzyme B (ImmunoGrB) to kill HER2-positive tumor cells. We constructed four ImmunoGrB fusion proteins containing different PEAII deletions and tested their abilities to kill HER2-positive cells. Our data showed that while a complete deletion of PEAII in ImmunoGrB resulted in an inability to kill cancer cells, ImmunoGrBs containing either PEAII (253-358aa) or PEAII (275-358aa) could efficiently kill HER2-positive SK-BR-3 cells. Most interestingly, the construct which contains only a furin cleavage site, named PEAII (275-280aa), could still induce SK-BR-3 apoptosis, although less efficiently. Moreover, delivery of the recombinant proteins by intramuscular plasmid injection led to an apparent tumor regression and prolonged animal survival in a nude mouse xenograft SK-BR-3 tumor model, indicating in vivo antitumor activity of the different PEAII containing ImmunoGrBs. Our results may help in understanding PEAII translocation and may lead to the development of useful tools or alternative therapy.