Defects or deficiencies in red cell membrane skeletal proteins often undermine the integrity and stability of the plasma membrane, and consequently cause hereditary hemolytic anemias. Genetic and biochemical studies have revealed a complicated picture of the organization of the membrane skeleton, within which α-/β-spectrin heterodimers form a protein lattice. By stabilizing the red cell membrane skeleton, the erythroid protein 4.1R greatly contributes to connecting and regulating the interaction among spectrins, actin filaments and integral proteins on the plasma membrane. In this study, we demonstrated the direct interaction between 4.1R and α-/β-spectrin. The results provide novel insights into the stoichiometry of 4.1R with spectrin, and demonstrate for the first time that the binding ratio of 4.1R to spectrin heterodimers is approximately 5.