This study investigates the spheroidization behavior of the O phase in Ti2AlNb alloy during high temperature deformation through a designed high-throughput experimental approach. The results of the high-throughput deformation experiments indicate that temperature, strain, and strain rate influence the spheroidization behavior of the O phase. Specifically, an increase in temperature and strain promotes the spheroidization of the O phase, while the strain rate exhibits the opposite effect. Moreover, the spheroidization mechanisms of this alloy during high-temperature deformation can be identified and primarily involve grain boundary separation mechanism, terminal dissolution mechanism, continuous dynamic recrystallization mechanism, edge spheroidization mechanism, and shear spheroidization mechanism. Furthermore, the analysis of experimental results reveals that the different morphologies of the spheroidized O phase have varying effects on the microscale mechanical response. In the region of large-sized high-density spheroidized O phase, the influence of back stress may extend to the entire B2 phase, thereby enhancing the B2 phase and subjecting the O phase and B2 phase to similar strains. Therefore, a small quantity of O phase is affected by the forward stress. Conversely, in the region of small-sized low-density spheroidized O phase, a small quantity of B2 phase is affected by the back stress, and the majority of the O phase is affected by forward stress. Eventually, the interaction mechanism between O phase and B2 phase during high-temperature deformation is explored for the first time through theoretical analysis.