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Transgenic plants that produce anti-insect substances are vital in improving crop yields and in reducing the environmental risks of chemical insecticides. Enhancin is a metalloprotease produced in occlusion bodies of the Trichoplusia ni granulovirus (TnGV). It is a key substance that enhances infection of the nucleopolyhedrovirus in lepidopteran insects. Rice (Oryza sativa L. cv. Nipponbare) protoplasts were cotransformed with pREXΦVEF and pLTHyg, which respectively bear the chimeric enhancin gene and the hygromycin-resistance gene. Hygromycin-resistant regeneration plants were examined by genomic polymerase chain reaction and genomic Southern and northern blotting analyses to confirm the presence and expression of the enhancin gene. Fourteen transgenic plant lines harboring the enhancin gene were obtained, and stable inheritance and expression of the enhancin gene were confirmed in the second, third, and fourth plant generations. Feeding Spodoptera exigua larvae leaves of enhancin-expressing rice plants in the presence of S. exigua nucleopolyhedrovirus occlusion bodies enhanced infection of the virus. Further, the development of Pseudaletia separata, S. exigua, and S. litura, none of which are host insects of TnGV, was inhibited when these larvae were fed enhancin-expressing rice leaves. This indicates that expression of the enhancin gene confers resistance to lepidopteran insect pests in rice.