In our previous research, we found that CYP6CY3 not only participates in the detoxification metabolism of neonicotinoid insecticides in cotton aphid but also affects their growth and development. However, how does transgenic cotton expressing dsAgCYP6CY3 affect the growth and development of cotton aphid? In this study, we combined transcriptome and metabolome to analyze how to inhibit the growth and development of cotton aphid treated with transgenic cotton expressing dsAgCYP6CY3-P1 (TG cotton). The results suggested that a total of 509 differentially expressed genes (DEGs) were identified based on the DESeq method, and a total of 431 differential metabolites (DAMs) were discovered using UPLC-MS in the metabolic analysis. Additionally, multiple DEGs and DAMs of glycolytic and The tricarboxylic acid (TCA) cycle pathways were significantly down-regulated. Pyruvate carboxylase (PC), citrate synthase (CS), malate dehydrogenase (MDH) enzyme activities and pyruvate content were reduced in cotton aphid treated with TG cotton. In addition, TG cotton could significantly decrease the total sugar content from the body and honeydew in cotton aphid. The above results indicated that TG cotton inhibited glycolysis and the TCA cycle, and this inhibition is consistent with previous studies showing that cotton aphid fed on TG cotton showed significantly reduced body length and weight as well as delayed molting. These findings provide a new strategy for reducing the transmission of viruses by cotton aphid honeydew, preventing fungal growth, mitigating impacts on normal photosynthesis and improving cotton quality.