20-Hydroxyecdysone (20E) and juvenile hormone (JH) control a variety of physiological events during insect development and metamorphosis. To understand how 20E and JH developmentally regulate energy metabolism in insects, we performed a genome-wide microarray analysis of fat body tissues isolated from the silkworm, Bombyx mori. Many genes involved in energy metabolism, including genes in the glycolytic pathway, were down-regulated during molting and pupation, when 20E levels are high. Notably, 20E treatment exhibited inhibitory effects on key glycolytic enzyme mRNA levels and activities, and RNA interference of the 20E receptor EcR-USP had the opposite effects to 20E treatment. Meanwhile, JH treatment stimulated both mRNA levels and activities of the key glycolytic enzymes, presumably via antagonizing the 20E action. Taken together, we conclude that 20E acts as a general blocker for glycolysis in the Bombyx fat body during molting and pupation, whereas the physiological role of JH is contrast with 20E during molting.