The effect of post-welding heat treatment (PWHT) on the microstructure and mechanical properties of large-thickness 2.25Cr1Mo0.25V steel was investigated through simulated post-welding heat treatment (SPWHT). The results showed that an increase in the SPWHT time decreased the toughness, hardness, and strength of the steel. After Min.SPWHT, the high-temperature tensile strength decreased more significantly, and the damage of Min.SPWHT to the high-temperature tensile strength reached approximately 80% of the Max.SPWHT. The microstructure of the tested steel before and after SPWHT consisted of granular bainite and lath bainite. After SPWHT, intergranular carbides were precipitated as coarsened carbides, carbide clusters, and chains of carbides; alloy element segregation occurred, and the segregation of Mo was the most serious, followed by Cr, and V. The precipitation behavior of the carbides and the increase in the effective grain size caused by the widening of the bainite–ferrite lath worked together and resulted in the decline of the impact toughness; the reduction in the solid solution and precipitation strengthening effects were the main factors in the strength reduction of the tested steel. In the high-temperature tensile tests, defects first appeared around the coarse carbides and carbide clusters. Controlling the size of the intergranular large-size carbides and the degree of cluster precipitation in the NT state structure may be a means of obtaining higher strength of the base metal subjected to PWHT.