Herein, the significant effects of rolling temperature and equivalent strain on the deformation microstructural evolution of a 304 stainless steel are characterized by X‐Ray diffraction, scanning electron microscopy, electron backscatter diffraction, and transmission electron microscopy, and the relationships between temperature, stacking fault energy (SFE), and deformation mechanisms are discussed. The SFE of experimental steel gradually increases from ≈19 to ≈48 mJ m−2 with increasing temperature from room temperature (20 °C) to 600 °C. A transition in deformation mechanisms occurs from martensitic transformation to deformation twinning and finally dislocation glide‐only, which is attributed to the increasing SFE caused by higher deformation temperature. Dislocation glide and deformation‐induced martensitic transformation dominate the plastic deformation during cold‐rolling at room temperature. Deformation twinning is observed in the higher temperature range where the SFE is between 28 and 38 mJ m−2, acting as a complementary deformation mechanism to dislocation glide. When the rolling temperature is increased to 600 °C, deformation twinning is inhibited. Dislocation slip becomes the sole deformation mechanism due to the high SFE. Meanwhile, the dislocation cells and highly dense dislocation walls are formed due to the high SFE.