Insects rely on innate immunity to defend against pathogens, with hemolymph playing a key role in immune responses. The silkworm (Bombyx mori) is a model organism for studying insect immunity, but research on immune-related proteins (especially antimicrobial peptides, AMPs) in silkworm pupae remains limited—despite pupae being rich in bioactive molecules with potential for antimicrobial development.To address this gap, we infected silkworm pupae with Bacillus subtilis (a Gram-positive bacterium) and used LC-MS/MS-based proteomics to analyze hemolymph proteins. The study aimed to identify immune-related proteins, clarify the pupal immune response dynamics, and provide insights for AMP discovery and application.