Cypermethrin (CYP) is a toxic manmade chemical compound belonging to pyrethroid insecticides contaminating the environment. Plantago major (PM) has numerous excellent advantages like high biomass yield and great stress tolerance, which make it able to increase the efficacy of phytoremediation. So far, no study has directly or indirectly made a transcriptome analysis (RNA-seq) of PM under CYP stress. The aim of this study is to identify the genes in PM related to CYP detoxification (10 μg mL−1) and compared with control. In this study, BGISEQ-500 high-throughput sequencing technology independently developed by BGI was used to sequence the transcriptome of P. major. Six libraries were constructed including (CK_1, CK_2, and CK_3) and (CYP_1, CYP_2, and CYP_3) were sequenced for transcripts involved in CYP detoxification. Our data showed that de novo assembly generated 138,806 unigenes with an average length of 1129 bp. Analyzing the annotation results of the KEGG database between the samples revealed 37,177 differentially expressed genes (DEGs), 18,062 down- and 19,115 upregulated under CYP treatment compared with control. A set of 107 genes of cytochrome P450 (Cyt P450), 43 genes of glutathione S-transferases (GST), 25 genes of glycosyltransferases (GTs), 113 genes from ABC transporters, 21 genes from multidrug and toxin efflux (MATE), 11 genes from oligopeptide transporter (OPT), and 3 genes of metallothioneins (MT) were upregulated notably. By using quantitative real-time PCR (qRT-PCR), the results of gene expression for 12 randomly selected DEGs were confirmed, showing the different patterns of response to CYP in PM tissues. Furthermore, the enzyme activity of Cyt P450 and GST in PM under CYP stress was significantly increased in roots and leaves than in control. This study introduces a clue to understand the metabolic pathways of plants used in phytoremediation by identifying the highly expressed genes related to phytoremediation which would be utilized to enhance pesticide detoxification and reduce pollution problem.