Objectives To identify an efficient in vitro refolding method to generate highly active His(6)-tagged scorpion toxin antitumor-analgesic peptide (AGAP) isolated from Escherichia coli inclusion bodies. Results N- and C-Terminal His(6)-tagged recombinant (r) AGAP (N-His6-rAGAP and C-His6-rAGAP, respectively) were expressed in E. coli; the purification and refolding conditions were optimized. C-His6-rAGAP, but not N-His6-rAGAP, exhibited significant in vitro antihepatoma activity that was much greater than that of rAGAP produced using SUMO fusion technology (IC50, 0.4 +/- A 0.08 vs. 1.8 +/- A 0.3 mu M). C-His6-rAGAP also showed significant inhibition of tumor growth in a mouse xenograft model of human hepatoma and inhibition of neuronal excitability, demonstrated by blockage of voltage-sensitive tetrodotoxin-resistant (TTX-R) sodium currents in acute isolated dorsal root ganglion neurons. Conclusions This refolding protocol optimized for C-terminal His(6)-tagged scorpion rAGAP is potentially applicable to similar long-chain and cysteine-rich toxins.