Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leadingto melanization.The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dualrole of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting andduring the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed inhemocytes and activated in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was activatedin the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reducedBmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticularmelanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, andproPO-activating enzyme. Our findings demonstrate that BmSPH-1 is responsible for the larval-pupal transformation, pupalcuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.