Abstract Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (∼92% purity) by cation-exchange chromatography and Ni–agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix™ Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1 M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18 h at 4 °C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC 50 of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 μM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.