Dislocation annihilation behavior of Sanicro 25 alloy was investigated based on various total strain amplitudes (Δεt/2). The slopes of cyclic stress response curves during stage ΙΙ were compared and showed that obvious stable cyclic behavior occurred at Δεt/2=0.3%. Microstructure analysis reveals that the number of slip plane increased with increasing Δεt/2. When Δεt/2=0.3%, dislocations can slip on the slip plane (111¯). When Δεt/2 increased to 0.4%, the slip planes included (1¯11) and (111¯). The fraction of substructured grains under Δεt/2=0.3% was obviously larger than that under Δεt/2=0.4%, which was attributed to the obvious dislocation annihilation. The deformed grains were dominant at Δεt/2=0.4%. The annihilation mechanisms of dislocations were analyzed during tensile and compressive loading based on the cyclic stress-time curves. The results showed that when Δεt/2=0.3%, during stage ΙΙ, dislocation annihilation mainly occurred during compressive loading. Dislocation annihilation included annihilation caused by cross-slip of screw dislocations and annihilation of adjacent edge dislocation dipoles. Furthermore, considering the critical annihilation distance between two adjacent edge dislocation dipoles, a modified dislocation annihilation model was presented. The dislocation annihilation model was verified based on the actual annihilation rate of dislocations. Further, the relationship of microstructure and fatigue properties was discussed. Based on the dislocation annihilation mechanisms, the fatigue life model considering dislocation annihilation was established. When the ratio of ρ¯− to ρ¯ increased, the fatigue life increased. Therefore, for cyclic hardening materials, obvious dislocation annihilation can increase fatigue life.