Elucidating the biosynthetic pathway of bioactive ingredients will provide an excellent opportunity for their sustainable supply. Phryma leptostachya, a traditional insecticidal plant, is rich in lignans, among which (+)-haedoxan A (HA) is the major insecticidal ingredient, but its biosynthetic pathway remains unclear. Bioassay results showed that HA exhibited remarkably high insecticidal activity against Mythimna separata with an LC50 value of 26.67 mg/L, which was higher than that of 3 common botanical insecticides celangulin-V, rotenone, and pyrethrin, and approximate to that of 3 common chemical insecticides indoxacarb, chlorantraniliprole, and permethrin. The main accumulation space of HA and two other representative lignans (+)-phrymarolin I and II was localized in the roots of P. leptostachya by using high performance liquid chromatography (HPLC) and bioassay methods. A comparative transcriptome analysis was applied to identify and characterize the full-length transcripts of root, leaf, and stem tissues using a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing, which provided a potential basis for further study the biosynthetic pathway of HA. This study is the first report on transcriptome analysis of P. leptostachya, and 13 isoforms probably involved in the HA biosynthesis were finally collected. In conclusion, these results contribute to further research into the biosynthetic pathway of HA and other bioactive lignans, and the transcriptome data provide a valuable resource for investigating this crucial insecticidal plant.