Cytokine-induced killer (CIK) cells, one of the feasible and effective methods of adoptive immunotherapy, have shown anti-leukemia activity in vivo and in vitro. But the strategy exhibits limited cytotoxic activity in clinical studies. In this study, CIK cells were transfected with an interleukin-3/Pseudomonas exotoxin gene (IL3PE38KDEL). RT-PCR and ELISA were used to verify the expression of IL3PE38KDEL in the transfected CIK cells. These cells released 1,186.7 ± 149.6 pg IL3PE38KDEL/10(4) cells over 48 h into the medium and the culture supernatant selectively killed IL3 receptor(IL3R)-positive HL60 cells, but not IL3R-negative K562 cells. Moreover, IL3PE38KDEL transfection did not influence phenotypes and cytokine production of CIK cells. Co-cultured with leukemia cells, IL3PE38KDEL transfected CIK cells showed enhanced cytotoxicity against IL3R-positive HL60 cells at all effector-to-target (E:T) ratios, but exerted a basal anti-leukemia activity against IL3R-negative K562 cells. Our findings demonstrate that IL3PE38KDEL gene transfection may be a novel strategy for improving anti-leukemia activity of CIK cells.