Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene.Because Cry1 Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops.The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments.The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin.In this paper, three media for crystal production and the method for Cry1 Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1 Ac protoxin.The results showed that the 1/2 LB(Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1 Ac protoxin in 300 m L 1/2 LB medium was(112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method.It will be a reference for other Cry protoxin preparation, especially for larger number. Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene. Baccident Cryl Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops. The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments. media for crystal production and the method for cryprotexin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin.In this paper, three media for crystal production and the method for Cry1 Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1 Ac protoxin. The results showed that the 1 / 2 LB (Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1 Ac protoxin in 300 m L 1/2 LB medium was (1 12.38 ± 5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method. It will be a reference for other Cry protoxin preparation, especially for larger number .