Diapause is a form of dormancy that organisms use to adapt to extreme environments by exhibiting developmental arrest. In the silkworm, Bombyx mori, diapause is thought to be elicited by diapause hormone (DH) signaling, which consists of interactions between DH and the DH receptor (DHR). However, the steps downstream of the DH signaling pathway are largely unknown. In the present study, we directly injected synthesized DH into the female pupae of a multivoltine, non-diapausing strain at 36 h after pupation. We found that the mRNA level of DHR declined at 4 h and recovered at 12 h after the injection of DH. Thus, we sequenced the transcriptome of the ovaries at 4 h and 12 h after the injection of DH. We identified 60 and 221 differentially expressed genes at 4 h and 12 h after the injection, respectively. All DEGs were identified, relating to 20E-related genes, JH-related genes, cellular detoxification, ribosomal proteins, lipid metabolism, and epigenetic modifications. Eleven genes were selected from the above categories to verify the transcriptome data. The qRT-PCR and RNA-Seq expression patterns of the genes were consistent, which indicated the authenticity and reliability of the transcriptome data. This study dramatically expands upon our knowledge of gene expression variation at the early phase of DH release.