In this study, the microstructural evolution and mechanical properties of B50A789G steels with different Nb/C ratios (7, 9, 11, and 13) after tempering at 495 °C were investigated through mechanical property testing, X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscope (TEM). The role of Nb in B50A789G steel was also explored. The results indicate that, at the same tempering time, the strength and hardness of the steel increase with increasing the Nb/C ratio. The maximum tensile strength exceeded 1240 MPa when the Nb/C ratio reached 13. With prolonged tempering time, the tensile strength of steels with low Nb/C ratios (7 and 9) gradually decreases, whereas steels with high Nb/C ratios (11 and 13) exhibit a decline in tensile strength only after 6 h of tempering. In contrast, the impact toughness shows an opposite trend to the strength. As the Nb/C ratio increases, both coarse primary NbC and nanoscale NbC precipitates in the steel gradually increase. The primary roles of Nb in B50A789G steel are grain refinement strengthening and precipitation strengthening. For steels with Nb/C ≤ 11, the improvement in strength is attributed to the combined effects of grain refinement strengthening and precipitation strengthening provided by Nb. However, for steels with Nb/C > 11, the increase in strength is primarily driven by the precipitation-strengthening effect of the nanoscale NbC phase.