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Abstract The oriental armyworm, Mythimna separata has led to great economic losses of corn in China. The main method to control this pest relies on chemical insecticides. Plenty of genes in armyworm can be activated in response to insecticides. In this study, the critical genes involved in detoxification of chlorantraniliprole in armyworm were investigated using RNA-seq (transcriptome sequencing), digital gene expression profiles, and functional identification by RNA interference (RNAi) technology. The de novo assembly of the transcriptomes was achieved based on Illumina short-read sequencing technology in M. separata. There were 70,845 unigenes produced and 35,327 (49.86%) annotated. 748 unigenes were significantly up-expressed and 741 were significantly down-expressed in chlorantraniliprole treatment group compared to the control group. Silencing of cytochrome B6-F (CYB6), and long chain fatty acid CoA ligase 4 (CoA) of the top 18 highly expressed genes selected in chlorantraniliprole treatment group, resulted in more significant sensibility of M. separata to chlorantraniliprole, growth retardation and antifeedant phenomenon, which indicated that both CYB6 and CoA were most possibly responsible for chlorantraniliprole metabolism/detoxication. [ABSTRACT FROM AUTHOR]