This present study aimed to clarify the effect of contact scale and surface topography of substrates with different roughnesses on the actual contact area, tangential stiffness, and tangential deformation of the substrate at micro- and macro-scales via finite element method (FEM) simulations, as well as the final tribological performances of MoS2 coatings by experiments. The MoS2 coatings were deposited on stainless steel (SS) substrates with different roughnesses, and the settings in the simulation models were based on the roughness of the SS substrates. The predicted tribological behavior of the simulation results was confirmed by the morphological and compositional analysis of the wear track using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), 3D profilometer, and Raman spectroscopy. The results showed that the substrate with a surface roughness of Ra 600 nm (R600), coated by MoS2 nanosheets, exhibited excellent tribological properties at both micro- and macro-scales. At the micro-scale, the lubrication lifetime of R600 was as long as 930 cycles, while the substrates with surface roughnesses of Ra 60 nm (R60) and Ra 6 nm (R6) had a lubrication lifetime of 290 cycles and 47 cycles, respectively. At the macro-scale, the lifetime of the substrate R600 was 9509 cycles, which was nearly six times longer than the 1616 cycles of substrate R60. For the rough surface of substrate, the surface grooves could not only effectively preserve the lubricant but also continuously release them, ensuring that the lubricants with low shear strength were always present in the contact interface. It was further verified that the high surface roughness of the substrate reduced friction and wear by reducing the actual contact area and enhancing the tangential stiffness of asperities, thereby prolonging the lubrication lifetime. The wear mechanisms were discussed in terms of the morphology and chemical composition of the wear tracks.