This study systematically investigates the interface structure of Nb films grown on MgO substrates with different orientations ((100) and (111)) by experiments and simulations. X-ray diffraction, transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) were used to characterize the structure of Nb films and the structure of interfaces between Nb films and MgO substrates. The results show that thin films exhibit different preferred planes on different orientations of MgO substrates. First-principles calculations were used to understand the interface configuration through a coherent interface model. The combination of experiments and simulations shows that the work of separation, together with substrate orientation and lattice mismatch, determines the interface structure between films and substrates.