The microstructural evolution, deformation mechanism and mechanical properties of Fe-25Mn-3Si-3Al-0.3Nb steel in the process of cold rolling were studied by optical microscopy, scanning microscopy, transmission electron microscopy, X-ray diffractometry, tensile testing and microhardness tests. A high-density dislocation structure and a small number of strain-induced twins appeared in the microstructure of the steel at 30% strain. At 50% strain, the strain-induced twins in austenite increased conspicuously, and the lamella thickness of the twins decreased. At 70% strain, the original grains were clearly refined by the micro-shear bands and twinning intersections to form a large number of sub-grains, and some sub-grains were at the nanoscale. The steel still remained a single-phase austenite during cold rolling even if the strain was as high as 70%. The plastic deformation mechanism of the steel was not changed through the addition of 0.3 wt.% Nb, and both dislocation slipping and twinning were still the fundamental plastic deformation mechanisms for the steel. Furthermore, cold rolling led to a drastic rise in the strength and hardness of the steel, but a remarkable decrease in the elongation. The characteristics of micropore aggregation fractures could always be observed on the fracture surface of static tensile specimens with various strains.