The retrogradation of wheat amylopectin during cold storage is the main reason for the increasing hardness of flour products such as steamed bread, bread and pastries, etc. Addition of gluten protein components is a green, safe, cheap and efficient method to inhibit the retrogradation of wheat amylopectin. In this paper, as being stored at 4 °C for 7 d, retrogradation rate of wheat amylopectin decreased from 55.02 % to 14.37 % after it was mixed with 20 % alkali-soluble glutenin (ASG) at 30 °C for 90 min, a 73.8 % reduction. The infrared results showed that the intensity of bending vibration of water molecules and intra-molecular β-sheet content of ASG decreased during the interaction between amylopectin and ASG. Meanwhile, intermolecular β-sheet and random coil contents of ASG increased. The results of 13C Solid-state NMR indicated that Qβ, Pγ and Lγ of ASG involved in interaction of wheat amylopectin, ASG and molecule of water. Under the optimal conditions, the interaction of wheat amylopectin and ASG began to form spheres containing disulfide bonds, resulting in the attenuation or disappearance of the diffraction peak at 2θ 19.7°, which may be marked as the criterion for the best mixing time of wheat amylopectin and ASG. The retrogradation kinetic index (n) of wheat amylopectin decreased significantly with the addition of ASG and formation of disulfide bond was the key factor. ASG could be potentially used as an anti-retrogradation agent for amylopectin.