The development of the dauer form of Caenorhabditis elegans daf-2(e1370) enhances the expression of genes such as fatty acid desaturase fat-6 and fat-7 and fatty acid elongase elo-2, and increases the level of triglyceride (TAG). RNA interference (RNAi) of the fat-6, fat-7, and elo-2 genes lowers fat accumulation in the nematode. We recently clarified the fact that RNAi of fat-related genes, especially fat-2, reduced fat accumulation and activated DAF-16. FAT-2 regulates the first step of polyunsaturated fatty acid (PUFA) synthesis. RNAi of fat-2 induced nuclear translocation of DAF-16 and increased the level of TAG that could be detected by Oil Red-O, but suppressed the accumulation of lipid dyed by Nile red. TAG levels are also increased in the adult daf-2(e1370), whereas Nile red staining showed fat reduction. Introduction of RNAi of fat-2, fat-6, fat-7, and elo-2 genes into the daf-16 deficient worm recovered Nile red-stained lipid storage. These results suggest that Nile red stained the lipids except TAG, and that the levels of these lipids are regulated by daf-16. In fat-2, fat-6, fat-7, and elo-2-RNAi worms, the Nile red-stained fat level was restored through addition of fatty acids, especially PUFA. This suggests that reduction of Nile red-dyed lipid reflects the disorder of fatty acid metabolism. Furthermore, treatment of the fat-2-RNAi worm with PUFA--using the fatty acids from linoleic acid through eicosapentaenoic acid--suppressed nuclear localization of DAF-16. These results suggest that PUFA acts as a mediator of daf-2/insulin signaling and that daf-16 might be involved in fatty acid homeostasis under the control of PUFA.