The microstructure, precipitates, and austenite grain in high-strength low-alloy steel were characterized by optical microscope, transmission electron microscope, and laser scanning confocal microscopy to investigate the effect of Mo on the toughness of steel. The microstructure was refined and the toughness was enhanced after the addition of 0.07% Mo in steel. The addition of Mo can suppress the Widmanstätten ferrite (WF) formation and promote the transformation of acicular ferrite (AF), leading to the fine transformed products in the heat-affected zone (HAZ). The chemical composition of precipitates changed from Nb(C, N) to (Nb, Mo)(C, N) because of the addition of Mo. The calculated lattice misfit between Nb(C, N) and ferrite was approximately 11.39%, while it was reduced to 5.40% for (Nb, Mo)(C, N), which significantly affected the size and number density of precipitates. A detailed analysis of the precipitates focusing on the chemical composition, size, and number density has been undertaken to understand the contribution of Mo on the improvement of steel toughness.