The Schmid factor (SF) is a critical parameter in crystal plasticity research that is often used to evaluate the level of difficulty in activating the slip systems within a grain. The evolution process and change mechanism of SF in 316 austenitic stainless steel during plastic deformation were investigated in this paper by using the in situ electron backscatter diffraction (EBSD) technique. The results showed that the average Schmid factor of global grains was highest in the original state, but after stretching, multiple rotation paths appeared in the grain, and the SF presented a monotonically decreased tendency with the increase in plastic strain degree. Numerical computation revealed that the decrease of SF was mainly governed by the change in φ angle, i.e., the angle between loading direction and slip plane normal increased inside the grains after the lattice rotation, which caused the slip plane to move parallel to the loading direction. The higher φ, the lower its cosine, which corresponds to low shear stress acting on the slip plane and could increase the difficulty of crystal slip.