After high-temperature forging and rapidly cooling, the microstructure evolution and spheroidization mechanism of powder metallurgy Ti-6Al-4 V alloy have been clarified. The spheroidization process is divided into two asynchronous stages. The initial stage is primarily characterized by the initial shearing and twisting of GBα grain boundaries, as well as the deformation of lamellar structure. Subsequently, the fragmentation of original GBα grain boundaries and the fracture of intra-granular α lamellae are observed. This process is accompanied by grain twisting and substructure formation, especially the division of lamellae through interface splitting and the segregation of local chemical elements. The differential accumulation of elements around the fractured and twisted grains suggests that element segregation and interfacial energy play a significant role in microstructure evolution. Therefore, the static spheroidization process largely depends on the formation and evolution of dislocation substructures, and the reduction of interfacial energy may be a key factor driving this process. These findings provide a theoretical basis for manufacturing metals with specific anisotropic properties by controlling microstructure evolution.