To explore the influence of rejuvenators on the relative molecular mass distribution and molecular size distribution of aged asphalt binders, the interaction between three rejuvenation agents and three long term aged asphalt binders was investigated. Gel permeation chromatography(GPC) was first used to detect the change of relative molecular mass distribution during rejuvenation process. Then the change of molecular size distribution was measured by calculating the relative proportion of the relaxation spectrum, which was derived from the dynamic shear testing of asphalt binders. The results show that the rejuvenators can both dissolve and dilute the large relative molecular mass and large size molecules in aged asphalt binder, and the effects depend on the type of both asphalt binder and rejuvenator. The connection between molecular size distribution and physical properties of asphalt binder indicates that the reduction of asphalt large size molecules has a strong relationship with the rejuvenation performance.