Large-diameter Ti6321 alloy seamless tube with Φ450 mm×20 mm was prepared using forging billet by cross piercing and hot rolling process, and the effect of annealing temperature on microstructure and mechanical properties of seamless tube were investigated. The results show that the microstructure of as-rolled tube is mainly composed of α phase and transformed β phase. Equiaxial structure is obtained after annealing at 940 ℃, duplex structure is obtained after annealing at 970 ℃, and Widmanstatten structure is obtained after annealing at 1020 ℃. With the increase in annealing temperature, the yield strength and tensile strength at room temperature of the tube are gradually decreased. The plasticity of the tube does not change much below the transformation point, but decreases sharply above the transformation point. The impact toughness first increases and then decreases. According to comprehensive analysis, the suitable annealing temperature for the prepared large-diameter Ti6321 alloy seamless tube is about 970 ℃, when the tube has the best impact performance and impact energy is 62 J. The average yield strength, tensile strength, and elongation of the tube after annealing at 970 ℃ are 786 MPa, 878 MPa, and 16.25%, respectively.