The difference of the transverse and longitudinal impact toughness of TC18 large-scale forging rods and its microstructural relationships have been systematically investigated in this study. Two types of impact samples, i.e., the C-L ones and the C-R ones, were machined from the head, middle and tail positions of the forging rods for Charpy impact testing. It is found that the impact toughness of the C-L samples is always higher than that of the C-R samples. The instrumented impact test further reveals that the ability to resist crack initiation is a key factor affecting impact toughness, whilst crack initiation energy of the C-L samples is significantly greater than that of the CR samples. Meanwhile, impact fracture observation manifests that cracks initiate in the form of micro-void coalescence, and mainly originate from the strong-hard phase (such as the α-phase of the grain boundary) near the notch of the samples. For the C-L samples, the elongated direction of the forging microstructure is parallel to the fracture direction, while it is perpendicular to the propagation direction after the micro-void initiation. The crack is thus difficult to grow to the critical size for unstable propagation, which results in the high consumed energy for crack initiation; for the C-R siblings, however, the elongated direction of the primary α-phase including grain boundary α-phase is parallel to the direction of crack initiation, and the cracks easily grow directly to the critical size along the strong-hard phase for unstable propagation, which causes a lower impact toughness in the samples.