The phase transformation and microstructure in Ti-22Al-25Nb alloy are extremely complex. In this work,the morphology evolution of the O phase during the heating and cooling process was investigated by electron backscatter diffraction(EBSD) and first-principles calculations. The results show that the O→αphase transformation process during the heating process is as follows: spheroidization of the O phase occurs first, then the αphase nucleates in the spheroidized O phase, grows and replaces the O phase,completing the O→αphase transformation. In the meanwhile, the diffusion of Nb from Nb-poor O to Nb-rich B2 phases is a back-diffusion process. According to first-principles calculations, the driving force of the O→αphase transformation is the difference in the free energies of formation for the two phases(0.09 eV/atom). When the Nb content is greater than 15.625 %, the lattice distortion of the αphase sharply increases, and the distortion energy drives the back-diffusion of Nb. During the cooling process,the α→O phase transformation is difficult and slow due to the difficult diffusion of Nb from the B2 to αphases. When holding for 60 min at 960℃, the coarse αphase gradually transforms to the O phase from the margin to the inside, forming a dispersed mixed structure of the O and αphases. During the B2→O transformation, the nucleation of the O phase induces a high stress region, in the range of approximately 200 nm.