Recently, infectious diseases have seriously inhibited the aquaculture of mud crab Scylla serrata in southeastern China. A better understanding of the immune molecules and defense mechanisms may be beneficial in reducing the harmful nature of these diseases. Available data show that hemocyanin (HMC) is a copper-containing respiratory protein present in the hemolymph of both mollusc and arthropod, and that it plays multiple roles in immune defense. In the current study, HMC from S. serrata (HMC-C) was isolated, and its hemolytic properties were investigated. The HMC-C shows hemolytic activities against vertebrate erythrocytes. The hemolysis displays dependency on pH, temperature, divalent cation, and HMC-C concentration. Complete hemolysis occurred at a concentration of 0.1 mg/mL, pH 5.0, and temperature of 37^°C in the presence of calcium. Furthermore, all 5 subunits of HMC-C were detected in the solubilized incubation products of erythrocytes with HMC-C, and the hemolysis could be inhibited to different degrees by osmoprotectants of various molecular masses. Together, our data suggest that HMC-C mediates hemolysis by inserting all 5 subunits into the erythrocyte membrane, causing cell rupture through a colloidosmotic mechanism.