As an insect-selective neurotoxin, scorpion long-chain Bj alpha IT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural Bj alpha IT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pastoris transformants showing high levels of secretory recombinant (r)Bj alpha IT. Secreted rBj alpha IT was expressed at levels as high as 340 mg/L following methanol induction in a fed-batch reactor, with similar to 21 mg of pure rBj alpha IT obtained from 200mL fed-batch culture supernatant by Ni2+-nitriloacetic acid affinity chromatography and CM Sepharose ion exchange chromatography. Injection of purified rBj alpha IT induced neurotoxicity symptoms in locust (Locusta migratoria) larvae, and the half-lethal dose of rBj alpha IT for locusts at 24-h post-injection ranged from 11 to 14 mu g/g body weight. These results demonstrated that large amounts of active rBj alpha IT were efficiently prepared from P. pastoris, suggesting this system as efficacious for determining rBj alpha IT insecticidal activity against other agricultural insect pests.