Overexpressing the Cry1Aa gene in sweetpotato significantly reduced pest damage through disrupting the integrity of the midgut of Spodoptera litura larvae for resistance against target Lepidoptera insect pests in sweetpotato. Sweetpotato is susceptible to insect pests and diseases leading to yield losses during pest outbreaks. Lepidoptera insects such as S litura are especially important pests of sweetpotato. The effect of Cry1Aa gene on S. litura was investigated by overexpressing Cry1Aa gene in sweetpotato to relieve symptoms due to pest damage. When transgenic leaves were fed to the larvae of S. litura, the growth of the larvae was reduced, the larval quality decreased, and mortality was increased compared with the larvae that fed on wild-type leaves. Further anatomical analysis revealed that the columnar cells of the midgut epithelium of the BT group were significantly damaged, loosened, or disordered. Furthermore, the integrity of the midgut was destroyed. In addition, when potted seedlings of the wild-type and BT sweetpotato were inoculated with the same number of S. litura larvae, wild-type plants died on the eighth day after infestation, while BT transgenic lines still grew normally. This study showed that transgenic sweetpotato overexpressing Cry1Aa can prevent S. litura infestation, and thus increase the yield of sweetpotato.