Abstract: Objective This study aimed to clarify the variation in impact toughness and its underlying micro-mechanisms in the heat-affected zone (HAZ) of DH36 thick plate steel under high heat input welding conditions. Methods Using a thermomechanical simulator, scanning electron microscopy, and in situ observation, the relationship between microstructure evolution and impact toughness in the HAZ of DH36 steel was systematically analyzed under varying heat inputs. Results Within the heat input range of 350～550 kJ/cm, the low-temperature impact energy of the HAZ first increased and then decreased. The optimum impact toughness was observed at 400 kJ/cm, with an impact energy of 235 J. The HAZ microstructure consisted primarily of polygonal ferrite, granular bainite, and minor pearlite. Bainitic ferrite nucleated at prior austenite grain boundaries and grew in a feather-like morphology into the grains, while acicular ferrite nucleated on intragranular precipitates and exhibited needle-like growth along specific crystallographic directions. Conclusion At a heat input of 400 kJ/cm, the HAZ exhibited numerous short and uniformly distributed bainitic ferrite laths along with an interlocking structure formed between acicular ferrite and bainitic ferrite. This cross-interlocked morphology was identified as the key factor contributing to the enhanced impact toughness.