High cycle fatigue failure has been recognized as one of the major forms of failure of aero-engine blades. This paper presents the high cycle fatigue testing of a Ni-based superalloy near orientation at 800 °C. The fracture morphology and dislocation configuration were analyzed in detail by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to indicate the influence of orientation deviation degree on the high cycle fatigue properties. The results show that the orientation deviation significantly affects the initiation of the slip systems, which is closely related to fatigue performance. The best fatigue life appears on the precise orientation, and the deformation behavior is controlled by multiple sets of equivalent {111} slip systems. With the increase in orientation deviation, the fatigue properties of the alloy degenerate significantly. On the boundary of -, two groups of {111} slip systems with the maximum Schmid shear stress dominate the deformation behavior. On the other hand, on the - boundary, the formation of stacking faults and rapid cutting of γ’ precipitates results in a negative effect on the fatigue life.