Many members of the Serine protease inhibitor (serpin) family participate in regulation by inhibiting Serine proteinases (SPs) or Serine protease homologs (SPHs) isozymes and maintaining the balance in the internal environment. SPs were expressed in the midguts of mid-fifth-instars and exhibited slightly higher expression in female compared to male late-fifth-instars. However, serpinsweremainly expressed in the silk glands, achieved a higher level of expression in the late fifth-instar and had an expression advantage in the males. The ratio of the cocoon shell was significantly higher in Jingsong compared to the other varieties in both the males and females, which was consistent with the higher mRNA and protein expression of Spi1 in the silk gland of Jingsong compared to the other varieties. These results reflected the positive correlation between TRP protein expression and the ratio of the cocoon shell and suggested that the higher mRNA and protein expression levels of the serpin gene wasmolecular evidence ofmore efficient silkmaterial synthesis. The SP/SPHand serpin genes presented different regulatory trends after treatment of the day 3 fifth-instar silkworm larvae with 20-hydroxyecdysone (20E) and juvenile hormone analogues (JHA). Themajority of the surveyed SP/SPH geneswere down-regulated and serpin geneswere up-regulated under treatmentwith the high concentration 20E. After JHA treatment, all of the investigated genes were up-regulated 48 h after treatment with JHA. The hormone regulatory effectwas primarily influenced by the developmental stage and the hormone dose. The tissue specificity and sex differences in the SP/ SPH and serpin genes implied that they participated in tissue-specific transcriptional regulation. The genes were expressed at different levels during different developmental periods and following hormone treatment. The results demonstrated that the genes played a role in scheduling and provided insights into the mechanismunderlying silk material formation and accumulation.