The effect of CaCl2 on denaturation and aggregation of silver carp myosin incubated at 40 °C was investigated by circular dichroism spectroscopy, surface hydrophobicity (S0-ANS), total sulfhydryl (SH) group content, zeta potential, turbidity, z-average diameter (dz), and dynamic rheological analysis. During setting at 40 °C, both CaCl2 and heating induced conformational changes of the fish myosin, and exposure of more hydrophobic amino acid residues and free SH groups, followed by myosin aggregation via hydrophobic interactions and disulfide bonds. Additionally, turbidity and dz of myosin increased significantly with increasing CaCl2 concentration, and the added CaCl2 further increased the extent and rate of aggregation of myosin by promoting the formation of Ca bridges. Myosin with 60 mM CaCl2 showed the maximal G' value and the highest rate of G' development. However, the G' value would decrease with an excessive amount of CaCl2 (100 mM).