Abstract: Ti6321 alloy is one of the most promising materials for structural components of unmanned equipment due to its excellent specific strength, corrosion resistance, and weldability, and the service conditions of the unmanned equipment impose even higher requirements on the impact resistance performance of Ti6321 alloy. However, the investigation on the relationship between the microstructure and impact toughness isn’t systematic and enough. In this work, instrumented impact tests are conducted to investigate the impact toughness of Ti6321 alloy with different microstructural characteristics. The results indicate that the impact toughness of Ti6321 alloy is primarily determined by crack propagation energy, which is significantly influenced by the orientation of α phase and volume fraction of primary and second α phases. When the α phase orientation is favored of the activation of prismatic slip sufficient dislocation and twinning deformation can effectively inhibit crack propagation. In this case, the impact absorbed energy of the duplex is higher than that of the basket-weave microstructure. Conversely, when the prismatic dislocation motion of the α phase is suppressed, the deformation compatibility between the primary α grains and the surrounding microstructure deteriorates, resulting in stress concentration, microcrack nucleation and crack propagation. Under this condition, reducing the volume fraction of primary α phase can enhance impact toughness. These findings suggest that hot working and heat treatment processes should be adjusted to control the orientations of the primary β phase, and primary and secondary α phases, and primary α volume fraction according to the service conditions to achieve the production of Ti6321 plate and other near-α alloys with superior impact resistances.