The steroid hormone 20-hydroxyecdysone (20E) triggers calcium signaling pathway to regulate 20E-response gene expression, but the mechanism underlying this process remains unclear. We propose that the 20E-induced phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) serves an important function in 20E-response gene transcription in the lepidopteran insect Helicoverpa armigera. CaMKII showed increased expression and phosphorylation during metamorphosis. 20E elevated CaMKII phosphorylation. However, the G-protein-coupled receptor (GPCR) and ryanodine receptor inhibitor suramin, the phospholipase C inhibitor U73122, and the inositol 1, 4, 5-triphosphate receptor inhibitor Xestospongin C suppressed 20E-induced CaMKII phosphorylation. Two ecdysone-responsible GPCRs and G-protein alpha q were involved in 20E-induced CaMKII phosphorylation by RNA interference analysis. 20E regulated CaMKII threonine phosphorylation at amino acid 290, thereby inducing CaMKII nuclear translocation. CaMKII knockdown by dsCaMKII injection into the larvae prevented the occurrence of larval-pupal transition and suppressed 20E-response gene expression. CaMKII phosphorylation and nuclear translocation maintained USP1 lysine acetylation at amino acid 303 by inducing histone deacetylase 3 phosphorylation and nuclear export. The lysine acetylation of USP1 was necessary for the interaction of USP1 with EcRB1 and their binding to the ecdysone response element. Results suggest that 20E (via GPCR activation and calcium signaling) activates CaMKII phosphorylation and nuclear translocation, which regulate USP1 lysine acetylation to form an EcRB1/USP1 complex for 20E-response gene transcription.