The nickel-based GH4169 superalloy is widely used in the aerospace field, which has desirable oxidation resistance and strength in extreme environments. However, the size effect and underlying microstructural mechanism in the cyclic deformation of the GH4169 sheet are still not fully elucidated, which affects the forming accuracy precision of thin-walled components. To this end, the cyclic mechanical properties and microstructural evolution of the GH4169 sheets with different thicknesses and grain sizes were systematically investigated via cyclic shearing tests. The superalloy sheet exhibits obvious early re-yielding, instantaneous softening and permanent softening during the cyclic deformation and these cyclic deformation behaviors are obviously affected by size effect. To rationalize the cyclic mechanical responses and size effect, transmission electron microscopy (TEM) and electron backscatter diffraction (EBSD) tests were conducted. The results indicated that the dislocation slip patterns are principally planar array slip and cross slip in the cyclic shearing deformation. Meanwhile, the annihilation of unstable dislocation and the presence of carbides led to the instantaneous softening. Moreover, the change of loading direction, the reduction of texture strength, the proportion of low-angle grain boundary (LAGB), and geometrically necessary dislocation (GND) density together result in the occurrence of permanent softening. Based on the comparative analysis of the microstructure evolution of superalloy sheets with different size factors, the underlying mechanism of size effect on cyclic deformation was clarified. The findings in the research combine the cyclic mechanical response and microstructure evolution closely in the cyclic deformation of superalloy sheets and deepen the understanding of size effect under complex loading paths.