Rheological properties, including high temperature rutting resistance and low temperature cracking resistance, of virgin, aged and rejuvenated asphalts were investigated by dynamic shear rheometer(DSR) and blending beam rheometer(BBR). Afterward, asphalt functional groups and microstructures of virgin, aged and rejuvenated asphalts were analyzed by means of Fourier transform infrared spectroscopy(FTIR) and atomic force microscopy(AFM) techniques. Results clearly demonstrate that rejuvenator could soften aged asphalt and improve its low temperature cracking resistance. Rejuvenator A is mainly composed of light components which are rich in aromatics, while rejuvenator B is found to be a complex organic mixture containing many polar functional groups. Moreover, rejuvenator B is able to chemically restore aged asphalt, however, there is just physically mixing between rejuvenator B and aged asphalt. There may be an interaction between aged asphalt and rejuvenator B, which depresses the vibrations of some specific functional groups of aged asphalt. Meanwhile, the aggregation of polar components after aging can be mitigated. After the addition of rejuvenator, the diffusion ability of molecules is enhanced, and the aggregation of polar components is stimulated, so bee structures grow larger and the number of microstructures decreases, the area ratio and height of bee structure are improved as well. The changes of these microstructures may be the intrinsic reasons for the change of rheological properties in the process of asphalt aging and regeneration.