Mitotic arrest deficient protein 2 (Mad2) is a typical metamorphic protein, which can adopt two distinct native folds at equilibrium under physiological conditions, an open inactive form (O-Mad2) and a closed active form (C-Mad2). This unusual two-state behavior of Mad2 and their interactions with cognate ligand Cdc20 plays a critical role in spindle assembly checkpoint signaling during mitosis. In this paper, interactions of O-Mad2 and C-Mad2 with TAMRA-Cdc20^121-138 were systematically investigated using fluorescence anisotropy techniques. As a result, the equilibrium dissociation constant of Mad2 two folds binding with Cdc20^121-138 were both within 10^-6 mol/L range in lower ionic strength solutions, and the K_D value of C-Mad2 and Cdc20^121-138 was 5 times lower than that of O-Mad2. While in high ionic strength solution, there was no obvious difference on KD value of C-Mad2 and O-Mad2 binding with Cdc20^121-138. The kinetic experiments suggested that the dissociation rate constant (k_d) between C-Mad2 and TAMRA-Cdc20^121-138 was similar to that of O-Mad2, but the association rate constant (k_a) between C-Mad2 and TAMRA-Cdc20^121-138 was one order of magnitude higher than that of O-Mad2, which suggested that the binding of C-Mad2 with Cdc20^121-138 is thermodynamically more stable and kinetically faster. Studies on interactions between Cdc20 mutants and Mad2 together with influence of ionic strength on their interactions both suggested that the interaction of Mad2 and Cdc20 is possibly not achieved by electrostatic interaction, but through hydrophobic interactions. Our results provide key information for revealing the conformational transition mechanism of metamorphic proteins and their important role in mitosis.