Abstract: It has been confirmed that hemocyanin (HMC) is a kind of immunoglobulin superfamily (IgSF) protein with antiviral and antibacterial activity. But until now, the molecular basis of its functional diversity is not well understood. In this study, four kinds of HMC fractions from Litopenaeus vannamei, i.e. A-HMCs, A-HMCl, AL-HMCs and AL-HMCl, were purified by affinity chromatography and lectin chromatography, which possessed distinct agglutinative activities against different pathogens. Among them, the agglutination activities of AL-HMCs and AL-HMCl were significantly (about 2–32 times) stronger than those of A-HMCs and A-HMCl against Escherichia coli K12 and Vibrio parahaemolyticus. Furthermore, the protein component and glycosylation modification of the 4 HMCs were analyzed by 2-DE and 2-D lectin blotting. The results showed that they had 6-7 spots in the 2-DE map with significant difference in isoelectric point and molecular weight. In particular, these protein spots could bind with 4 lectins, including concanavalin A (ConA), peanut agglutinin (PNA), ulex europaeus agglutinin (UEA) and dolichos bifows agglutinin (DBA), to different degrees. Of these, all of the 6 protein spots from A-HMCl could bind to 4 kinds of lectins, while only 4 and 3 protein spots from A-HMCs showed positive to UEA and DBA, respectively. AL-HMCs and AL-HMCl could respectively react with PNA and UEA, to produce 3 spots out of 7 total protein spots and 2 spots out of 6 total protein spots, respectively. Collectively, our data demonstrated that the molecular basis of functional diversity of shrimp hemocyanin may be closely related to the diversity of protein composition and glycosylation.