Large diameter Ti6321 alloy seamless tube with φ450*20mm was prepared using forging billet by cross piercing and hot rolling process, and the effect of annealing temperature on the microstructure and mechanical properties of seamless tube were investigated. The results show that the as-rolled tube microstructure is mainly composed of α phase and transformation β, equiaxial structure is obtained after annealing at 940℃, duplex structure is obtained after annealing at 970℃, widmanstatten structure obtained after annealing at 1020℃; With the annealing temperature increasing, the room temperature yield strength and tensile strength of the pipe will gradually decrease. The plasticity of the pipe will not change much below the transformation point, but will decrease sharply above the transformation point; the impact toughness first increased and then decreased. Comprehensive analysis shows that the suitable annealing temperature of the prepared large diameter Ti6321 seamless alloy pipe is about 970℃, and the pipe has the best impact performance and impact energy is 62J.In addition, the yield strength, tensile strength and elongation of the pipe after annealing at 970℃ are 786MPa, 878MPa and 16.25%, respectively.