Juvenile hormone (JH) and 20-hydroxyecdysone (20E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant (Met) and germ-cell expressed (Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C (PKC) which phosphorylated ultraspiracle (USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. The usp^S35A mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20E signaling that delayed developmental timing. The usp^S35A mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.