The influence of strain amplitude and fatigue cycles on cyclic deformation of polycrystalline specimens of commercially 99.53% purity nickel was investigated using fully reversed strain-controlled fatigue tests. The fatigue tests were performed under constant strain amplitude, in air and at room temperature, at a constant strain rate of 0.0001 s−1. The objective was to study cyclic deformation behavior and characterize cyclic hardening response by microstructural observations using transmission electron microscopy. The cyclic stress–strain (CSS) curve of cyclically deformed polycrystalline nickel exhibited three distinct regions with a short quasi-plateau region in the intermediate amplitude range and a slight increase of saturation stress with plastic strain amplitude. The plastic strain amplitude at which the test was conducted influences the cyclic hardening rate. Dislocation structures in fatigued polycrystalline nickel are amplitude dependent and are classified into three types of dislocation structures corresponding to the three regions in the CSS curve. Vein structures consisting of loop patches were observed at low strain amplitudes. These structures become mixed with labyrinth structures at intermediate amplitudes. In addition, persistent slip bands (PSBs) were observed in the quasi-plateau regions of CSS curve. Cellular structures were observed at higher amplitudes which become increasingly equiaxed and smaller with fatigue cycles. A correlation between stress–strain response and development of dislocation structures in deformed polycrystalline nickel revealed that the saturation stress is linearly related to the inverse wall spacing. This relationship is equivalent to the mesh-length theory of work hardening over the strain range used in this study.