AISI 321 stainless steel has excellent resistance to intergranular corrosion and is generally used in nuclear power reactor vessels and other components. The as-cast and wrought structures are quite different in hot workability, so physical simulation, electron back-scatter diffraction, and hot processing maps were used to study the mechanical behavior and microstructure evolution of as-cast nuclear grade 321 stainless steel in the temperature range of 900–1200 °C and strain rate range of 0.01–10 s⁻¹. The results showed that the flow curve presented work-hardening characteristics. The activation energy was calculated as 478 kJ/mol. The fraction of dynamic recrystallization (DRX) increased with increasing deformation temperature and decreasing strain rate. DRX grain size decreased with increasing Z value. Combining the hot working map and DRX state map, the suggested hot working window was 1000–1200 °C and 0.01–0.1 s⁻¹. The main form of instability was necklace DRX. The nucleation mechanism of DRX was the migration of subgrains. The δ phase reduced the activation energy and promoted DRX nucleation of the tested steel.