Pheromone biosynthesis-activating neuropeptide (PBAN), produced in subesophageal ganglion, is known to stimulate pheromone biosynthesis in Plutella xylostella. The pheromone production is more active in the scotophase than in the photophase, which suggests that there may be changes of gene expression in the pheromone glands. To analyze gene expression involving in pheromone biosynthesis for 24 hrs, we performed transcriptomes of pheromone glands which were isolated at every 4 h. Eleven pheromone biosynthesis-related genes, acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, Δ9 desaturase, Δ11 desaturase, fatty acid reductase (FAR), alcohol oxidase, aldehyde oxidase, and aldehyde reductase were identified. Among these genes, ACC, FAS and chain shortening enzymes involving in early stage of pheromone biosynthesis exhibited their highest expression between AM9 and PM5. Desaturases, Δ9 and Δ11, showed the peak of expression at PM1 and AM5 or PM5, respectively. Interestingly, FAR expression was the highest at AM1, active reproductive period. These results suggest that genes involving in pheromone biosynthesis can be sequentially regulated for their biological roles.