Rolling–sliding wear experiments of U75V rail steel were carried out on a GPM-40 friction and wear machine. The microstructure evolution and property change of the sample under rolling–sliding conditions were studied using scanning electron microscopy (SEM), electron backscatter diffraction (EBSD), and hardness test. In the early wear stage, the wear mechanism of the sample surface is mainly oxidative wear, and the sample is in uniform wear stage. With the increase in cycle number, the surface wear mechanism changes to fatigue wear, the mass wearing quantity increases significantly, while the wear rate remains unchanged after increasing; thus, the sample is in the rapid wear stage. As the slip ratio increases, the mass wearing quantity and the wear rate increase. During the plastic deformation of U75V rail steel, cementite is bent, fractured, fragmented, and even dissolved, and the proportion of high-angle boundary (HAB) in ferrite gradually increases. The increase in slip ratio accelerates the microstructure refinement of U75V rail steel. As a result, the proportion of high-angle boundary increases obviously at the same distance from the surface, and the grain refinement becomes serious. The maximum hardness is located at the worn surface. With the increase in cycle number, the hardness of the worn surface increases rapidly at first, reaches the maximum, and then reaches saturation. The saturation hardness is about 850 HV, increasing by 1.38 times. The increase in slip ratio improves the hardening speed of U75V rail steel but exerts a minimal effect on the saturation hardness. Surface hardening results from the combination of dislocation strengthening, strengthening by grain size reduction, and solid solution strengthening.