Symmetrical push-pull low-cycle fatigue (LCF) tests were performed on Ti-2Al-2.5Zr samples with different grain sizes (5 and 40 μm) at room temperature (RT) and low temperature (77 K). The results show that the coarse-grained samples of 40 μm exhibit a higher ductility and LCF life than the fine-grained ones at RT. Meanwhile, the fine-grained samples of 5 μm displayed improved ductility and LCF life at 77 K compared with those at RT. Microstructural observations using optical microscopy (OM), scanning electron microscopy, and transmission electron microscopy (TEM) revealed that a transition occurred in the plastic deformation mode, from twinning with slip to slip alone, as the grain size decreased from 40 to 5 μm at RT. Conversely, in the fine-grained samples fatigued at 77 K, twinning was activated and became one of the dominant plastic deformation modes. The improvement in the LCF life of the coarse-grained samples at RT and the fine-grained ones at 77 K could be attributed to the activation of deformation twinning. In addition, the cyclic stress response curves showed that cyclic stress saturation was exhibited in the fine-grained samples at all strain ranges. An initial cyclic hardening followed by cyclic softening was displayed ahead of the cyclic stress saturation in coarse-grained samples at high strain amplitudes. When the testing temperature decreased to 77 K, cyclic stress hardening prior to cyclic stress saturation also appeared in the fine-grained samples. The relation among the grain size, testing temperature, plastic deformation modes, and LCF life in Ti-2Al-2.5Zr was subsequently discussed.