To elucidate the molecular mechanisms of underlying diapause in the larvae of Ostrinia furnacalis, we aimed to identify potential diapause-associated genes and signaling pathways. We employed the Illumina NovaSeq^TM 6000 high-throughput sequencing platform to sequence the O. furnacalis larvae in three states: non-diapause (ND), diapause (D), and diapause-termination (DT). Subsequent bioinformatics analysis and diapause-related genes screening were conducted. The transcriptomic sequencing results were validated using qRT-PCR. Comparisons between D vs ND and DT vs D yielded 3, 520 and 1, 107 differentially expressed genes (DEGs), respectively. Functional enrichment analysis of DEGs in the D vs ND group primarily enriched pathways related to the proteasome, amino acid and nucleotide sugar metabolism, protein processing in the endoplasmic reticulum, and tyrosine metabolism. In contrast, the DT vs D group showed significant enrichment in pathways associated with the biosynthesis of valine, leucine, and isoleucine; oxidative phosphorylation; glycine, serine, and threonine metabolism; and other polysaccharide degradation and nitrogen metabolism pathways. qPCR validation of nine randomly selected DEGs corroborated the transcriptomic sequencing results. Furthermore, we identified seven ecdysteroid-related genes, twenty juvenile hormone-related genes, eight circadian rhythm signaling pathway-related genes, twenty-one Ca²⁺ signaling pathway-related genes, and five insulin-related genes that were hypothesized to play crucial roles in the regulation of diapause in O. furnacalis larvae, which provided essential groundwork for the identification of molecular targets for the eco-friendly control of O. furnacalis.