Abstract: Previous studies have demonstrated antibacterial and antifungal activities of hemolymph-derived hemocyanin upon pathogen infection with unknown mechanisms. To investigate how peptides derive from hemocyanin, multiple methods including size-exclusion chromatography, Tricine-SDS-PAGE, immunoblotting, bacterial agglutinative and antibacterial assays were applied. The results showed that shrimp Litopenaeus vannamei hemocyanin could produce seven peptides via trypsin digestion, ranging from 6 to 70 kD, which could be identified by rabbit anti-shrimp hemocyanin antibody specifically. These peptides aggregated with Vibrio parahaemolyticus in vitro, whose agglutinative activity was 4—16 folds higher than that of full length hemocyanin. The peptide 3 possessed obvious antibacterial activities against V. parahaemolyticus with a antibacterial rate (93.76±1.60)% at the concentration of 75 μg/mL, which was significantly higher than that of the control group (P<0.01). In addition, N-terminal Edman Sequencing analysis showed that the peptide 3 was located in the α-helix region of N-terminus of shrimp L. vannamei hemocyanin small subunit. These discoveries will help to understand how the hemocyanin derived peptides are formed and to establish effective strategies for shrimp disease control.