Abstract: To investigate the differences in fatigue behavior of welded joints of 2.25Cr1Mo0.25V steel for hydrogenation reactors, low-cycle fatigue tests were conducted at 450 °C under various strain amplitudes. Scanning electron microscopy and electron backscatter diffraction were used to characterize the microstructures, and the microstructure evolution and failure mechanisms of weld metal in different states under thermo-mechanical interaction were revealed. The results show that under the condition of cyclic loading at 450 °C, the temperature provides thermal energy for alloying elements, while the stress during cyclic loading supplies corresponding driving force for the diffusion of alloying elements. This causes the granular bainite in the as-welded (AW) specimen to completely transform into ferrite, whereas carbide precipitation is promoted at 450 °C for the specimen with post-weld heat treatment at 705 °C for 8 h. The metastable phases in the specimen completely decompose, and carbides grow along grain boundaries in a chain-like distribution, resulting in stress concentration near the grain boundaries. Meanwhile, under the thermo-mechanical coupling, atomic diffusion and grain boundary migration are promoted, leading to a decrease in the number of grains with low grain orientation spread (GOS) values, a more uniform distribution of grain orientation, and higher Schmidt factor (SF) values. As the number of cycles increases, the cyclic stress of the AW specimen shows a rapid softening phenomenon at strain amplitudes of 0.6% and 0.7%, while the cyclic stress of the PWHT specimen decreases steadily.