Abstract Dislocation structure and its impact on the mechanical behavior of a γ′ strengthened nickel base single crystal alloy under high cycle fatigue (HCF) at 870 °C was studied. The results indicate that HCF lifetime declines with increase of applied stress amplitude. In the early stages, the fatigue deformation occurs by forced bowing of dislocations through the narrow γ matrix channels on {111} planes. During mid-term fatigue stages, most of the dislocations formed in the matrix are located in the γ/γ′ interfaces, whose intersection and reaction produce new dislocation segments and three-dimensional dislocation networks. The Burgers vectors of the dislocations in the network are 1/2 . The interaction of cyclic stress with high temperature induces the precipitation of homogeneous globular γ′ particles, which is beneficial to fatigue strength. At the end of fatigue test, the cyclic stress leads to the formation of persistent slip bands moving through the γ matrix channels and γ′ particles. Dislocation shearing through the γ′ phase is found occasionally. The extrinsic stacking faults is also observed.