The influence of the degree of plastic deformation by cold rolling on the microstructure and mechanical properties of high-nitrogen austenitic steel VNS-53-Sh‑M is studied by scanning and transmission electron microscopy. It is shown that under the experimental deformation conditions the initial austenitic grains are stretched in the rolling direction. The packets of microtwins and increased density of dislocation substructures are formed inside the grains. At a rolling strain of 20%, these packets are observed along several twinning systems. When the deformation degree is increased up to 60%, the formation of deformation localization bands of the shear band type is detected. The intersection of microtwins in different crystallographic orientations with shear bands leads to the formation of submicrocrystalline scale fragments. The changes of microstructure under plastic deformation conditions causes an increase in strength and a decrease in plastic properties compared to those in the quenched state. At room tensile test temperature, the yield strength increases by a factor of 1.5 and 1.9 relative to the quenched state at the strain rates of 20 and 60%, respectively. At the same time, the elongation to fracture remains at a fairly good level within 7.4–12.6%.