Polymer chain ends play an important role in the glassy dynamics of polymeric materials. In this study, a combination of single molecule defocus fluorescence microscopy and well-controlled atom transfer radical polymerization was used to investigate site-dependent segmental mobility of poly(n-butyl methacrylate). As the temperature increased, the rotation of fluorophores, which were selectively labelled in chain end and chain middle, was gradually activated. The power spectra of rotation trajectories, the distribution of angular displacement as well as the population of rotating fluorophores demonstrated that the local dynamics was more activated at the chain ends than the middles, showing the unique contribution of the chain end to the dynamics of the system. Polymer chain ends play an important role in the glassy dynamics of polymeric materials. In this study, a combination of single molecule defocus fluorescence microscopy and well-controlled atom transfer radical polymerization was used to investigate site-dependent segmental mobility of poly (n-butyl As the temperature increased, the rotation of fluorophores, which were selectively labeled in chain end and chain middle, was gradually activated. The power spectra of rotation trajectories, the distribution of angular displacement as well as the population of rotating fluorophores said that that the local dynamics was more activated at the chain ends than the middles, showing the unique contribution of the chain end to the dynamics of the system.