Introduction:Insect cytochrome P450 (CYP450) genes play important roles in the detoxification and metabolism of xenobiotics, such as plant allelochemicals, mycotoxins and pesticides. The polyphagousConogethes punctiferalisis a serious economic pest of fruit trees and agricultural crops, and it shows high adaptability to different living environments.Methods:The two novel P450 genesCYP6CV1andCYP6AB51were identified and characterized. Quantitative real-time PCR (qRT-PCR) technology was used to study the expression patterns of the two target genes in different larval developmental stages and tissues ofC. punctiferalis. Furthermore, RNA interference (RNAi) technology was used to study the potential functions of the two P450 genes by treating RNAi-silenced larvae with three commonly used pesticides.Results:TheCYP6CV1andCYP6AB51genes were expressed throughout variousC. punctiferalislarval stages and in different tissues. Their expression levels increased along with larval development, and expression levels of the two target genes in the midgut were significantly higher than in other tissues. The toxicity bioassay results showed that the LC50values of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin onC. punctiferalislarvae were 0.2028 μg/g, 0.0683 μg/g and 0.6110 mg/L, respectively. After treating with different concentrations of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin (LC10, LC30, LC50), independently, the relative expressions of the two genesCYP6CV1andCYP6AB51were significantly induced. After the dsRNA injection, the expression profiles of the two CYP genes were reduced 72.91% and 70.94%, respectively, and the mortality rates of the larvae significantly increased when treated with the three insecticides independently at LC10values.Discussion:In the summary, after interfering with theCYP6CV1andCYP6AB51inC. punctiferalis, respectively, the sensitivity ofC. punctiferalisto chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin was significantly increased, indicating that the two CYP6 genes were responsible for the adaptability ofC. punctiferalisto the three chemical insecticides inC. punctiferalis. The results from this study demonstrated thatCYP6CV1andCYP6AB51inC. punctiferalisplay crucial roles in the detoxification of chlorantraniliprole, emamectin benzoate and lambda-cyhalothrin.