The effect of annealing temperature on the microstructures and mechanical properties of the cold-rolled Fe-25Mn-3Al-3Si TWIP was studied by means of electron backscatter diffraction technology, transmission electron microscope and tensile test. The Hall-Petch relationship was analyzed in detail. The results have shown that the microstructure of the fully recrystallized samples consists of equiaxed grains and annealing twins. With the increase of the annealing temperature, the grain size is continuously increasing while the fraction of Σ3 grain boundaries is fluctuantly increasing. After annealed at 850 ℃ for 1 h, the Σ3 grain boundaries reach 44%. In the tensile process, the relationship of strength and grain size obeys the Hall-Petch relationship, but twin boundaries affect the slope K of the Hall-Petch relationship. The K(ε)-ε relationship of the TWIP steel is different from that of general steels at room temperature, in which K(ε) monotonically increases with the strain ε. The K(ε) of the TWIP steel increases first, then, keeps unchanged and finally decreases with the strain ε.