Abstract: The pearlite microstructure exhibits excellent tribological properties due to its unique layered structure, which allows the synergistic effect between ferrite and cementite. However, due to technical limitations, the mechanical origin of the superior tribological properties of pearlite is still not fully understood, especially in terms of the effects of layer direction and layer thickness on tribological behavior of pearlite. In this study, the effects of layer direction and layer thickness on the nano-scratching tribological behavior of pearlite were investigated by molecular dynamics simulation. The results indicated that compared with the layer direction parallel to the surface, the layer direction perpendicular to the surface performs better tribological behavior with a smaller depth of the plastic affected region and a fewer number of the wear atoms. For pearlite with layer direction perpendicular to the surface, the thickness of the layer had a little effect on the depth of the plastic affected region, but the friction force, friction coefficient and number of the wear atoms decreased with the decrease of the layer thickness. In particular, the thickness of the layer was closely related to the morphology of the pile-up. This work contributed to the understanding of the relationship between the layer direction and the layer thickness of pearlite and the microscopic mechanism of tribological deformation, which provided the theoretical guidance for designing pearlitic steel with outstanding tribological properties.