Niobium (Nb) is mainly added to low-carbon steels, in which the main microstructure is ferrite and/or bainite, to refine grains and improve their properties. However, the studies on Nb in high-carbon pearlitic steels are insufficient, and there are some debates regarding the existing studies. High-carbon pearlitic steel is widely used in bars, wires, and steel rails. The role of Nb in high-carbon steels may differ for different steel grades and under different heat treatments. Hence, further detailed studies are required to improve the properties of high-carbon pearlitic steels. In this study, two series of high-carbon pearlitic steels (68C and 75C series) were designed and subjected to isothermal pearlite transformation at different austenitization and isothermal transformation temperatures. The microstructures and mechanical properties of the steels were investigated by optical microscopy, field-emission scanning electron microscopy, electron backscatter diffraction, scanning transmission electron microscopy, and hardness tests. The results showed that the pearlite lamellae were refined by the addition of Nb to high-carbon pearlitic steels, which was attributed to the solute drag effect of Nb. The pearlite colony and nodule sizes also became finer with the addition of Nb because of the finer prior austenite grains and smaller pearlite growth rates. However, the refinement of the pearlite lamellae, nodules, and colonies by the addition of Nb was not significantly enhanced by increasing the Nb content from 0.014 wt% to 0.027 wt% in 75C series samples. In addition, the pearlite interlamellar spacing and colony and nodule sizes of the samples decreased with decreasing isothermal and austenitization temperatures. Moreover, the addition of Nb to high-carbon steels increased their hardness and yield strength. The main strengthening mechanisms of Nb in high-carbon steel are grain refinement strengthening and precipitation strengthening in the samples austenitized at 900 °C, whereas the improvement of strength is mainly attributed to grain refinement strengthening in the samples austenitized at 1180 °C. This study systematically characterized the pearlite morphology of Nb microalloyed high-carbon steels and analyzed the strengthening mechanism, providing a theoretical reference for the development of Nb-containing high-carbon pearlitic steels.