The microstructure and toughness of tungsten inert gas (TIG) backing weld parts in low-pressure steam turbine welded rotors contribute significantly to the total toughness of the weld metal. In this study, the microstructure evolution and toughness of TIG weld metal of 25Cr2Ni2MoV steel low-pressure steam turbine welded rotor under different post-weld heat treatment (PWHT) conditions are investigated. The fractography and microstructure of weld metal after PWHT are characterized by optical microscope, SEM, and TEM, respectively. The Charpy impact test is carried out to evaluate the toughness of the weld. The optical microscope and SEM results indicate that the as-welded sample is composed of granular bainite, acicular ferrite and blocky martensite/austenite (M-A) constituent. After PWHT at 580 °C, the blocky M-A decomposes into ferrite and carbides. Both the number and size of precipitated carbides increase with holding time. The impact test results show that the toughness decreases dramatically after PWHT and further decreases with holding time at 580 °C. The precipitated carbides are identified as M23C6 carbides by TEM, which leads to the dramatic decrease in the toughness of TIG weld metal of 25Cr2Ni2MoV steel.