BACKGROUND: ⊍-linolenic acid is an essential unsaturated fatty acid in organisms. However, there is a large gap between ⊍-linolenic acid accumulation and its synthesis mechanism in insects. Fatty acid synthases (FASs) and desaturases (Desats) are vital enzymes required for the synthesis of unsaturated fatty acids.RESULTS: The pupae of Glyphodes pyloalis (Lepidoptera: Pyralidae), which is a destructive pest of mulberry trees, contain the highest level of ⊍-linolenic acid compared to other life-history stages. To further explore the synthesis mechanism of ⊍-linolenic acid in G. pyloalis pupae, we constructed a pupal transcriptome dataset and identified 106 genes related to fatty acid metabolism from it. Among these, two fatty acid synthases (GpylFAS) and five desaturases (GpylDesat) were identified. A qRT-PCR validation revealed that GpylFAS1 and GpylDesat1, 2, 3, 5 were expressed highest at pupal stages. Furthermore, the content of ⊍-linolenic acid decreased significantly after silencing GpylFAS1 and GpylDesat5, respectively. Besides, knocking down Gpyl-FAS1 or GpylDesat5 resulted in more malformed pupae and adults, as well as lower emergence rates. Meanwhile, silencing Gpyl-FAS1 or GpylDesat5 affected the expressions of the other GpylFASs and GpylDesats.CONCLUSION: The present results illustrate the pivotal function of FASs and Desats in ⊍-linolenic acid biosynthesis and metamorphosis in insects. Our research also broadens the sources of unsaturated fatty acids, especially for ⊍-linolenic acid from insects, and provides novel insights for the management of mulberry insect pests from the perspective of utilization rather than control.