The striped rice-stem borer, Chilo suppressalis (Walker) is one of the most important pest of rice worldwide. In this study, a proteinaceous extract from Citrullus colocynthis L. shows various degrees of inhibition to digestive α-amylases of C. suppressalis. Digestive α-amylases of larvae were inhibited by different concentrations (approximately 50%) of C. colocynthis amylase inhibitor (CCAI). One of the isozymes totally disappeared and the sharpness of another decreased on native-PAGE electrophoresis. The pH dependency of inhibition revealed that the enzyme was inhibited in a wide range of pH from 7–10, and the optimal pH of the enzyme occurred in lepidopteran larvae. The highest inhibition of α-amylase by CCAI was observed at 25°C; the temperature near the optimal temperature of amylolytic activity. A time-course experiment demonstrated that enzymatic activity was the highest, 30 min after the onset of the experiment, when the highest inhibition occurred. The enzyme kinetic studies using Lineweaver-Burk analysis, revealed a mixed inhibition of CCAI on α-amylase activity. The current study is the first basic one using α-amylase inhibitor against C. suppressalis. This study opens the way for transgenic rice varieties containing inhibitors.