By means of appropriate cold rolling pre-deformation, the tensile strength and plasticity of a novel polycrystalline precipitation-strengthened Nickel-based superalloy with low-stacking fault energy at 750 °C can be enhanced. The findings indicate that such pre-deformation leads to the emergence of microscopic substructures, including dislocations, anti-phase boundaries (APBs), stacking faults, and Lomer-Cottrell Locks (L-C locks). It is observed that the presence of these substructures is increased with the degree of pre-deformation. At the 15 % pre-deformation, a few deformation twins are detected near the grain boundaries, and their occurrence rises further in specimens subjected to 25 % pre-deformation. In addition, the nano-twins formation presents a strong correlation with the intensification of Brass-type textures. Notably, after the cold rolling pre-deformation, the alloy's yield strength as well as tensile strength at 750 °C is enhanced in contrast to those without pre-deformation, which is attributed to plentiful factors, such as low recovery and recrystallization activation energy, high-density nano-twins, grain refinement, and conducive crystallographic orientation for nano-twin mechanism activation caused by cold rolling pre-deformation. The specimen with 25 % pre-deformation exhibits relatively high tensile ductility. In this study, the relationships between pre-cold rolling deformation, microstructure, texture, and mechanical properties are comprehensively explored, indicating that the cold rolling pre-deformation holds promise for enhancing Nickel-based superalloys' strength and plasticity.