The mechanical properties of ASTM F1684 (Invar 36) alloy manufactured via laser beam powder-bed-fusion (PBF-LB) technique are strongly influenced by the non-equilibrium solidification microstructure. Due to the complexity of printed microstructure, e.g., the presence of melted pools, nano-precipitates, and high-density dislocations, it is very challenging to disentangle the specific mechanisms responsible for the fatigue properties. Here, a comparative investigation of the fatigue properties between PBF-LB and conventionally manufactured (CM) samples was conducted. Dedicated characterizations, including electron channeling contrast imaging (ECCI), and high-angular resolution EBSD (HR-EBSD), were performed. The mid-cycle (104–105 cycles) fatigue behaviors of both samples are comparable, while the fatigue resistance of PBF-LB sample becomes inferior at high-cycle range (≥106 cycles). This disparity can be ascribed to the competing effect between crack nucleation and propagation. PBF-LB samples are more susceptible to fatigue crack initiation due to the printed macro-defects. Whereas, the homogeneous plastic deformation and the grain fragmentation, both triggered by the ultrafine cellular structure, efficiently mitigate crack propagation rates. Based on these findings, the current understanding of the underlying mechanisms governing the fatigue performance of PBF-LB Invar 36 alloy is deepened, emphasizing the significant advantages of non-equilibrium solidification microstructure in retarding fatigue crack propagation.