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• GNS layer induced by SMRT enhanced the strength-ductility synergy of 316L steel. • Gradient microstructure effectively inhibits strain localization during deformation. • SMRT 316L displays phase transformation and cyclic softening under fatigue at 650 ℃. • Fatigue life is well predicted by CPFE model with plastic-slip-based indicator. • Multi-scale failure mechanism is studied for GNS 316L under strain fatigue at 650 ℃. Gradient nanostructured materials have excellent fatigue resistance. At present, there are few research on the fatigue properties of gradient nanostructured (GNS) materials at high temperature. Herein, the multi-scale failure mechanism of GNS 316L steel at 650 °C is studied, and the high temperature fatigue tests under different strain amplitudes are conducted. Then, post-test microstructure observations were carried out to reveal the damage mechanisms, and the fatigue behavior was numerically simulated by crystal plasticity model. The experimental results show that the strength of the GNS 316L steel is still higher than that of the coarse-grained (CG) 316L steel at 650 °C, and the fatigue life of the GNS 316L steel is also higher than that of the CG one under strain fatigue loadings. The fatigue fracture results show that the fatigue crack presents a mixed propagation mode, and the crack source of GNS 316L transfers from surface to subsurface under low strain amplitude. The numerical results directly reflect the influence of the GNS surface layer on the plastic slip behavior. The fatigue failure mechanism of GNS 316L steel under strain-controlled cyclic loading at 650 °C was explained from a multi-scale perspective. This study provides data and method support for the application of GNS 316L steel at high temperature. [ABSTRACT FROM AUTHOR]