Abstract: Nitrogen, as an efficient and economical alloying element, is one of the element to regulate the microstructural stability and mechanical properties of duplex stainless steels. However, studies on the influence of nitrogen content variation on the low-cycle fatigue life of S32750 duplex stainless steel and the micro-deformation mechanisms of the two phases under cyclic loading are still relatively insufficient. In this regard, three experimental steels with different nitrogen contents were designed and prepared with nitrogen content as the core variable. Tensile tests and low-cycle fatigue tests were carried out to systematically study the effects of nitrogen content on the strength and fatigue properties of the experimental steels. Thermo-Calc calculations, optical microscopy (OM) and transmission electron microscopy (TEM) were combined to characterize the microstructure and the evolution of dislocation morphologies of two phases in the experimental steels. The results show that the strength of experimental steels is co-affected by multiple factors including nitrogen solid-solution strengthening, heat treatment temperature and ferrite content. Under the synergistic effect of multiple factors, the yield strength difference of experimental steels with nitrogen mass fraction ranging from 0.23% to 0.34% is controlled within 10 MPa. The low-cycle fatigue life of experimental steel with 0.23% nitrogen mass fraction is about 1.5 times that of experimental steels with 0.29% and 0.34% nitrogen mass fraction. The main reason is that the increase in nitrogen content raises the stacking-fault energy and intensifies the lattice distortion of austenite, which promotes the formation of dislocation pile-up and deformation twins. The deformation structure of the austenite phase gradually evolves from a single slip band to a composite structure of slip bands, microbands and deformation twins. In experimental steels with high nitrogen content (0.29% and 0.34% mass fraction), the massive formation of deformation twins in austenite accelerates local plastic dissipation, which is more likely to induce fatigue crack initiation and eventually lead to fracture failure. This study clarifies the influence of the nitrogen content on the low-cycle fatigue properties and micro-deformation mechanisms of S32750 duplex stainless steel, which can provide a theoretical basis for composition optimization and process design of high-life duplex stainless steels.