In this paper, EBSD, SEM, TEM and quasi-static uniaxial tensile tests were used to study the effects of temperature and twin boundaries on the mechanical properties and Protevin-Le chatelier（PLC) effect of GH3625 alloy. The results show that with the increase of annealing temperature (1000 °C~1160 °C), the recrystallized grains of the annealed specimen grow, resulting in the annihilation of part of the annealed twin boundaries, which reduces the content and strength of the annealed twin boundaries of the alloy. And it is found that the lower the annealing temperature, the greater the critical strain value of the alloy with PLC. At the same time, the tensile deformation of the annealed specimen at 25 °C and 290 °C is mainly dominated by dislocation slip and deformation twin, while at 565 °C, the dislocation slip is mainly dominated by dislocation slip, and a large number of lamination faults are formed. With the increase of deformation temperature, dislocations tend to pass through grain boundaries and annealed twin boundaries, resulting in PLC, weakened grain boundary strengthening effect, and decreased alloy strength. In addition, the specimen with an annealing temperature of 1000 °C has an excellent combination of strength and plasticity in the temperature range of 25 °C~565 °C, which is mainly attributed to the interaction of pre-existing fine grains and a large number of annealed twin boundaries with dislocations, as well as the combined effect of deformed twins and lamination faults formed during tensile deformation. In addition, it is found that the twin content and deformation temperature have a significant effect on the rheological amplitude of the PLC.