Abstract: The microstructure evolution and performance characteristics of the heat-affected zone (HAZ) in laser welding of 10Ni5CrMoV steel are investigated. By designing novel thermal simulation specimens and utilizing a thermal simulation testing machine, the thermal cycling behavior during the laser welding process was simulated. The peak temperatures (T_m) were 1 350 °C, 1 000 °C, and 765 °C, respectively, with t_8/5 times of 1.5 s, 2.5 s, and 5.0 s. Various characterization methods, including impact testing, tensile testing, metallographic analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electron backscatter diffraction (EBSD), were employed. The results indicate that when T_m is 1 350 °C and 1 000 °C, respectively, the primary microstructure under different t8/5 times is lath martensite, and the grain size significantly increases with the increase of the peak temperature. When T_m = 1 350 °C and t_8/5 = 1.5 s, the impact energy significantly decreases to 43 J. The analysis suggests that large carbide particles are formed between martensite laths, which leads to a decrease in material toughness. However, at the same peak temperature, when t_8/5 is extended to 2.5 s, the impact energy is comparable to that of the base metal. This is primarily attributed to the synergistic effect of lath martensite and retained austenite, which significantly improves the material toughness.