The high-strength bolts of grid structures with bolted spherical joints under the action of suspension cranes are at risk of severe fatigue failure. Thus, this paper studies the variable-amplitude fatigue performance of M60 high-strength bolts. The test results for eight specimens in four loading modes are obtained using an Amsler fatigue testing machine. The fatigue life is also estimated based on Miner and Corten–Dolan’s theories, and the applicability of Corten–Dolan’s theory is verified. The fracture induced by the variable-amplitude fatigue is microscopically analyzed using scanning electron microscopy (SEM), revealing the mechanism of the variable-amplitude fatigue failure. Our findings provide valuable experimental data supporting the fatigue life estimation of grid structures with bolted spherical joints in service.