The fatigue fracture mechanism of a nickel-based single crystal (NBSC) superalloy with recrystallized grains was studied at 550 °C by in situ observation with a scanning electron microscope (SEM) for the first time. Multiple crack initiations associated with recrystallized grain boundaries and carbides were observed. By analysis of the slip traces and crack propagation planes, the operated slip systems were identified to be octahedral for both single crystal substrate and recrystallized grains. Distinct crystallographic fractures dominated, accompanied by recrystallized grain boundary associated crack initiations. This is different from the widely reported solely intergranular cracking at high temperature. Fatigue crack growth rate curves showed evident fluctuation, due to the interaction of fatigue cracks with local microstructures and the crack coalescence mechanism. Both the recrystallized grains and the competition between different slip systems were responsible for the deceleration and acceleration of fatigue microstructurally small crack behavior.