Abstract Diapause is a quintessential survival strategy orchestrated through animal–environment coevolution. Although extensive studies have been conducted using the silkworm, Bombyx mori , as the predominant diapause model, the molecular mechanisms underlying this process remain largely elusive. In this study, through transcriptomic profiling of serosal tissues comparing diapause‐destined (DD) and nondiapause‐destined silkworm eggs, we identified 2 790 upregulated and 2 791 downregulated genes in DD serosa. Functional enrichment analysis revealed significant associations of differentially expressed genes with key metabolic pathways, including amino sugar and nucleotide sugar metabolism, cell cycle regulation, and fatty acid elongation. Temporal expression profiling demonstrated stage‐specific expression patterns of these differentially expressed genes across different diapause phases. CRISPR/Cas9‐mediated knockout of the DD serosa‐specific trehalose transporter gene ( Tret1 ) reduced the trehalose consumption, concomitant with reduced sorbitol accumulation, and thus decreased the diapause incidence. Our findings provide novel insights into the serosa‐mediated regulatory mechanisms governing embryonic diapause in B. mori .