Diapause is an adaptive strategy characterized by the arrest of growth or reproductive development, which enables insects to survive from adverse living environment. The diapause induction process in the silkworm, a typical embryonic diapause insect, has been extensively studied. However, the molecular mechanisms governing the release of the diapause hormone (DH) during pupal stage remain unclear. The diapause characteristics of bivoltine silkworms are transgenerationally induced as a maternal effect. When eggs are incubated at 25℃, DH is released from the suboesophageal ganglion (SG) of female pupa, resulting in the production of diapause eggs by adult moths. In contrast, incubation at 15℃ suppresses DH release, leading to the laying of non-diapause eggs. In this study, the eggs of silkworm were incubated at 25℃ and 15℃ respectively. During the critical period of DH release (day 3 of the pupal stage), brain-suboesophageal ganglion (Br-SG) complexes were dissected from female pupae for transcriptome sequencing. Comparative analysis revealed 888 differentially expressed genes (DEGs). Among these, 568 genes were up-regulated and 320 genes were down-regulated in the 15℃ incubation group relative to the 25℃ incubation group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were predominantly associated with molecular functions such as serine-type endopeptidase activity, monooxygenase activity, GTPase activation activity and G protein-coupled receptor activity. Additionally, the DEGs were found to be primarily enriched in pathways related to hormone biosynthesis, terpenoid skeleton biosynthesis, longevity regulation pathway-multi-species, neural activity ligand-receptor interaction and other related pathways. This study identifies key target genes and establishes a foundational framework for elucidating the molecular mechanism underlying DH release during the pupal stage in the diapause induction process of silkworm.