The insect steroid hormone 20E-hydroxyecdysone (20E) regulates gene transcription via a genomic pathway by forming a transcription complex that binds to DNA with the help of the chaperone proteins Hsc70 and Hsp90. However, the non-genomic mechanisms by which 20E regulates gene expression remain unclear. In this study, we found that 20E regulated the phosphorylation of serine/threonine protein kinase cyclin-dependent kinase 10 (CDK10) through a non-genomic pathway to mediate gene transcription in the lepidopteran Helicoverpa armigera. The downregulation of CDK10 by RNA interference in larvae and the epidermal cell line delayed development and suppressed 20E-induced gene transcription. CDK10 was localized to the nucleus via its KKRR motif, and this nuclear localization and the ATPase motif were necessary for the efficient expression of 20E-inducible gene. The rapid phosphorylation of CDK10 was induced by 20E, whereas it was repressed by the inhibitors of G-protein-coupled receptors, phospholipase C, and Ca2+ channels. Phosphorylated CDK10 exhibited increased interactions with heat shock proteins (Hsps) Hsc70 and Hsp90 and then promoted the interactions between Hsps and ecdysone receptor EcRB1 and the binding of Hsps-EcRB1 complex to the 20E response element for the regulation of gene transcription. CDK10 depletion suppressed the formation of the Hsps-EcRB1 complex at HR3 promoter. These results suggest that 20E induces CDK10 phosphorylation via a non-genomic pathway to regulate gene transcription in the nucleus.