In this study, a series of experiments including stress relaxation, nanoindentation, tension loading experiments, and transmission electron microscope (TEM) examination were performed to investigate microstructural evolution, the stress relaxation behavior, and the influences of stress relaxation and high-cycle fatigue on mechanical properties of high strength cable. The stress relaxation experiments were performed on a single steel wire for 1000 hours at three initial stress levels to characterize the stress relaxation behavior of the tested cable. Microstructural evolution of the specimens deformed by stress relaxation was observed and analyzed using TEM examination. Long-term relaxation behavior was then calculated and investigated. Finally, the influences of stress relaxation and high-cycle fatigue on material properties were studied by performing nanoindentation and tension loading experiments on the deformed relaxation specimens. The degradation of mechanical properties under stress relaxation was interpreted through a micro-mechanism regarding the microstructural evolution and the grain boundary strengthening. The results from the present study can be used for practical designs as well as for assessing the stress relaxation behavior and fatigue resistance of high-strength cables.