In this paper, a novel electromagnetic negative-stiffness spring (ENSS) is constructed by coaxial permanent annular magnets and coils with rectangular cross section. The stiffness of the ENSS is negative when the coils carry exciting current. In the stiffness characterization study, the analytical model of the ENSS based on filament method is established and validated experimentally. With the stiffness model, the factors affecting the electromagnetic force and stiffness characteristics are investigated in a quantitative way. The effectiveness of a vibration isolator based on the ENSS is tested on a prototype isolator. The relationships between the transmissibility and the exciting frequency are measured under diverse amount of exciting currents. The experiment results demonstrate that the performance of the isolator can be easily tuned online.