Update publication information. Juvenile hormone (JH) antagonizes 20-hydroxyecdysone (20E) signaling mainly through the intracellular receptors Met and Gce in Drosophila. 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. Importantly, JH was identified to phosphorylate USP at Ser35, the PKC phosphorylation site required for the maximal action of 20E through its nuclear receptor complex EcR-USP. Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated PKC to phosphorylate USP at Ser35. The USPS35A mutant, in which Ser35 was replaced with Ala35 by genome editing, showed attenuated 20E signaling and Yorkie activity, resulting in delayed developmental timing and reduced body size, respectively. Moreover, genetic interaction experiments confirmed that JH lied upstream of PKC-mediated USP phosphorylation at Ser35. This study propose a novel model of JH membrane signaling that exhibits crosstalk with different pathways simultaneously to regulate distinct developmental processes.