Insect herbivores adapt and develop strategies to counteract plant chemical defenses. The aphid Uroleucon formosanum is a serious sap‐sucking pest that infests lettuces containing toxic sesquiterpene lactones (STLs). Herein, we employed a combination of genome sequencing and RNA‐seq transcriptome profiling to understand the mechanisms underlying phytotoxin tolerance in U. formosanum. We generated the first chromosome‐level genome assembly for U. formosanum, with a total size of 453.26 Mb and a scaffold N50 of 33.22 Mb. Comparative genomic analyses revealed an enrichment of signals for positive selection and gene family expansion in immune‐related pathways. Specifically, the expanded set of heat shock protein 70 (HSP70) genes showed upregulation after treatment with lactucin, suggesting that they may play a role in the immune response against STLs. The expression of takeout‐like genes and cuticle‐associated genes was also significantly increased in the lactucin‐treated samples. Additionally, 53 cytochrome P450 monooxygenase, 30 carboxylesterase, 19 glutathione S‐transferase, 32 uridine diphosphate glycosyltransferase and 63 ATP‐binding cassette (ABC) transporter genes were identified in the U. formosanum genome. CYP4C1, CYP6A13 and 7 ABC genes were strongly upregulated in response to lactucin treatment, indicating the involvement of detoxifying enzymes in the tolerance of U. formosanum to STLs. Our findings suggest that the cuticle barrier, immune response and enzyme‐mediated metabolic detoxification jointly enhance the tolerance of U. formosanum to phytotoxins and promote its adaptation to host plants. This study presents a valuable genomic resource and provides insights into insect adaptation to plant chemical challenges and future technological developments for pest management.