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High-cycle fatigue (HCF) tests at different temperatures were performed on a single crystal superalloy SRR99 with [001] orientation. The results demonstrate that conditional fatigue strength firstly increases and then decreases with the increase of temperature. It exhibits the same tendency with tensile strength at elevated temperatures. The microstructures are observed by SEM and TEM and it is found that the morphology of g′ particles changes significantly and dissolution of g′ particles takes place during cyclic loading. This is likely induced by the back and forth movement of interface dislocations during the cyclic loading. As a result, the strengthening effect from the coherent g/g′ coherent interfaces is gradually deteriorated during high-cycle fatigue deformation. On the other hand, the fatigue crack propagation is found to be primarily along a specific crystalline plane, which is identified as (111). The specific mechanism for the microstructure evolution during cyclic loading is discussed based on SEM and TEM observations.